Digital Collections > USF Electronic Theses and Dissertations > Microglia activation in Alzheimer's disease\nTownsend, Kirk Phillip\nDissertations, Academic -- Pathology and Laboratory Medicine -- Doctoral -- USF\nDissertations, Academic -- Pathology and Laboratory Medicine -- Doctoral -- USF ( lcsh )\nABSTRACT: The work detailed in this dissertation has an overarching theme of modulating microglia activation in both in vivo and in vitro models relevant to AD. The premise is that understanding microglia function in this context may lead to a better understanding of AD pathogenesis and thus to effective therapeutic interventions. In chapter 3, we employ a well-defined model of microglia activation whereby the intraperitoneal delivery of LPS results in CNS microglia activation and TNF-α production. Having previously identified that CD45 signaling pathways antagonized microglia TNF-α production in vitro and given that immunotherapy with anti-CD45 antibodies are already in clinical trials for both the treatment of malignant disorders as well as for tolerance induction following organ transplants, we investigated whether microglial CD45 could be a relevant molecular target in the opposition of microglia activation in vivo.Given that a number of epidemiological studies have shown an inverse correlation between the use of statins (a class of drugs that were initially described as specific inhibitors of cholesterol biosynthesis) and the incidence of Alzheimer's disease (Wolozin, 2000; Simon 2002; Zamrini, 2004); in chapter 4, we employed one of the most prescribed statins, lovastatin in our microglia activation paradigm. Interestingly, we show that statins only inhibit the enzyme HMG-CoA (the rate limiting step of cholesterol biosynthesis) but that they also display pleiotrophic effects including the inhibition of CD40 expression. Given the role of CD40 in microglia activation and its potential role in AD pathogenesis, we investigated whether lovastatin's protection in AD might be derived from effects on microglia function as governed by the CD40 pathway.In chapter 5, we provide evident for a model of microglia activation that addresses some of the current controversies concerning the role of microglia cells in AD pathogenesis. The model suggests that microglia cells exist in a number of distinct activation states, in one such state that we denote the "phagocytic state"; microglia function to clear cellular debris or foreign invaders or in the case of AD to remove β-amyloid/Aβ peptides. However, in response to certain co-stimuli (i.e. CD40 activation) these microglia take the form of an "antigen presenting cell" (APC) whereby they lose their phagocytic capacity in lieu of cytokine production and thus potential contributing to AD pathogenesis.\nby Kirk Phillip Townsend.\n001498257 ( ALEPH )\n57709300 ( OCLC )\nAJU6862 ( NOTIS )\nE14-SFE0000489 ( USFLDC DOI )\ne14.489 ( USFLDC Handle )\ncontrolfield tag 001 001498257\nsubfield code a E14-SFE0000489\nTownsend, Kirk Phillip.\nMicroglia activation in Alzheimer's disease\nABSTRACT: The work detailed in this dissertation has an overarching theme of modulating microglia activation in both in vivo and in vitro models relevant to AD. The premise is that understanding microglia function in this context may lead to a better understanding of AD pathogenesis and thus to effective therapeutic interventions. In chapter 3, we employ a well-defined model of microglia activation whereby the intraperitoneal delivery of LPS results in CNS microglia activation and TNF-α production. Having previously identified that CD45 signaling pathways antagonized microglia TNF-α production in vitro and given that immunotherapy with anti-CD45 antibodies are already in clinical trials for both the treatment of malignant disorders as well as for tolerance induction following organ transplants, we investigated whether microglial CD45 could be a relevant molecular target in the opposition of microglia activation in vivo.Given that a number of epidemiological studies have shown an inverse correlation between the use of statins (a class of drugs that were initially described as specific inhibitors of cholesterol biosynthesis) and the incidence of Alzheimer's disease (Wolozin, 2000; Simon 2002; Zamrini, 2004); in chapter 4, we employed one of the most prescribed statins, lovastatin in our microglia activation paradigm. Interestingly, we show that statins only inhibit the enzyme HMG-CoA (the rate limiting step of cholesterol biosynthesis) but that they also display pleiotrophic effects including the inhibition of CD40 expression. Given the role of CD40 in microglia activation and its potential role in AD pathogenesis, we investigated whether lovastatin's protection in AD might be derived from effects on microglia function as governed by the CD40 pathway.In chapter 5, we provide evident for a model of microglia activation that addresses some of the current controversies concerning the role of microglia cells in AD pathogenesis. The model suggests that microglia cells exist in a number of distinct activation states, in one such state that we denote the "phagocytic state"; microglia function to clear cellular debris or foreign invaders or in the case of AD to remove β-amyloid/Aβ peptides. However, in response to certain co-stimuli (i.e. CD40 activation) these microglia take the form of an "antigen presenting cell" (APC) whereby they lose their phagocytic capacity in lieu of cytokine production and thus potential contributing to AD pathogenesis.\nAdviser: Paul R. Sanberg.\nCo-adviser: Jin Q. Cheng.\nx Pathology and Laboratory Medicine\nM I C R O G L I A A C T I V A T I O N I N A L Z H E I M E R S D I S E A S E by K I R K P H I L L I P T O W N S E N D A di s s e r t a t i on s ubm i t t e d i n pa r t i a l f u l f i l l m e nt of t he r e qui r e m e nt s f or t he de g r e e of D oc t or of P hi l os ophy D e pa r t m e nt of P a t hol ogy C ol l e ge of M e di c i ne U ni ve r s i t y of S ou t h F l o r i da M a j or P r of e s s or : P a ul R S a nbe r g, P h. D D S c C o M a j or P r of e s s or : J i n Q C he ng, P h. D M D J un T a n, P h. D M D P a ul a B i c kf or d, P h. D H unt i ngt on P ot t e r P h. D D a t e of A ppr ova l : N ove m be r 8 2004 K e yw or ds : A C D 45, S t a t i n pha goc yt os i s C opyr i ght 2004 K i r k P h i l l i p T ow ns e nd\nA C K N O W L E D G E M E N T S I w a nt t o f i r s t t ha nk e a c h m e m be r of m y di s s e r t a t i o n c om m i t t e e : m a j or pr o f e s s or D r P a ul R S a nbe r g ; c o m a j o r pr o f e s s or D r J i n Q C he ng; m e m be r s ; D r J un T a n, D r P a ul a B i c kf or d a nd D r H unt i ng t on P ot t e r f or t he i r s t e a df a s t s uppor t a nd e nc our a ge m e nt dur i ng m y P hD s t udi e s I a m e s pe c i a l l y i nde bt e d t o D r J un T a n w i t hou t w hos e m e nt or s hi p t h i s di s s e r t a t i on w oul d not ha ve be e n r e a l i z e d. F i na l l y I w oul d l i ke t o t ha nk a l l t he i ndi vi dua l s w i t h w hom I ha ve ha d t he pl e a s ur e t o e xc ha nge i de a s a nd w ho e i t he r di r e c t l y or i ndi r e c t l y m a de t hi s P hD pos s i bl e e s pe c i al l y : D e m i a n O br e gon, M a r t i na V e ndr a m e a nd N a n S un.\ni T A B L E O F C O N T E N T S L I S T O F T A B L E S vi L I S T O F F I G U R E S vi i A B S T R A C T i x C H A P T E R O N E I N T R O D U C T I O N 1 1. 1 E pi de m i ol ogy o f A l z he i m e r s di s e a s e 1 1. 2 S i gn a nd S ym p t om s 2 1. 3 D i a gnos i s 3 1. 4 T r e a t m e nt 4 1. 4. 1 M a na ge m e nt of C ogni t i on 4 1. 4. 2 M a na ge m e nt of N on c ogni t i ve S ym pt o m s 6 1. 5 H a l l m a r k A l z he i m e r s di s e a s e pa t hol ogy 7 1. 5. 1 A m yl oi d 7 1. 5. 2 N e ur of i br i l l a r y T a ngl e s 8 1. 5. 3 I nf l a m m a t i on a nd G l i os i s 9 C H A P T E R T W O G E N E R A L M A T E R I A L S A N D M E T H O D S 11 2. 1 P e pt i de s a nd d r ugs 11 2. 1. 1 C y3 A pe pt i de p r e pa r a t i on 11 2. 1. 2 A nt i bodi e s a nd r e c om bi na nt pr o t e i ns 11 2. 2 M ur i ne p r i m a r y c e l l c ul t ur e s 12 2. 3 M i c r ogl i a l pha goc yt os i s a s s a y of A 13 2. 3. 1 U s i ng F I T C A 13 2. 3. 2 U s i ng C y3 A 14 2. 3. 3 V i a A i m m unopr e c i pi t a t i on a nd i m m unobl ot 15\ni i 2. 4 E L I S A m e a s ur e of c yt oki ne a na l ys i s 16 2 5 M i c r ogl i a A nt i ge n P r e s e nt a t i on A s s a y 16 2 6 C D 40 de t e c t i on 17 2. 6. 1 C D 40 m R N A de t e c t i on by R T P C R 17 2. 6. 2 F l ow c yt om e t r y a na l ys i s of c e l l s ur f a c e C D 40 18 2. 6. 3 W e s t e r n bl ot a na l ys i s of C D 40 pr ot e i n 19 2. 7 I m m unobl ot a na l ys i s of J A K / S T A T pa t hw a y 20 2. 8 S t a t i s t i c a l A na l ys i s 21 C H A P T E R T H R E E C D 45 A S A M O L E C U L A R T A R G E T T O O P P O S E L P S I N D U C E D M I C R O G L I A L I N F L A M M A T I O N 22 3. 1 I nt r oduc t i on 22 3. 2 R e s ul t s 24 3. 3. 1 C D 45R B i s t he m os t pr e va l e nt C D 45 i s of or m e xpr e s s e d on m i c r ogl i a l c e l l s 24 3. 3. 2 L i ga t i on of C D 45R b a nt a goni z e s L P S i nduc e d m i c r ogl i a l a c t i va t i on i n v i t r o 25 3. 3. 3 I n v i v o c r os s l i nki ng C D 45R B i nhi bi t s br a i n T N F pr oduc t i on 25 3. 3 D i s c us s i on 28 C H A P T E R F O U R L O V A S T A T I N I N H I B I T S I F N # I N D U C E D C D 40 A C T I V A T I O N 33 4. 1 I nt r oduc t i on 33 4. 2 R e s ul t s 35 4. 2. 1 L ova s t a t i n b l oc ks I F N # i nduc t i on of C D 40 e xpr e s s i on a s s e e n by R e ve r s e T r a ns c r i pt a s e P ol ym e r a s e C ha i n R e a c t i on, W e s t e r n B l ot t i ng a nd F l ow c yt om e t r y a n a l ys i s 35 4. 2. 2 L ova s t a t i o bl oc ks C D 40 l i ga t i on i nduc e d T N F I l 6, I L 1 p r oduc t i on a s de t e r m i ne d by E nz ym e L i nke d I m m unoa ds or ba nt A s s a y. 37 4. 2. 3 L ova s t a t i n P r om ot e s m i c r ogl i a l pha goc yt i c f unc t i o n w hi l e a nt a goni z i ng C D 40L i nduc e d m i c r ogi a l A P C f unc t i on. 40 4. 3 D i s c us s i on 48\ni i i C H A P T E R F I V E M I C R O G L I A L A N T I G E N P R E S E N T I N G C E L L F U N C T I O N I S I N D U C E D B Y C D 40 L I G A T I O N 53 5. 1 I nt r oduc t i on 53 5. 2 R e s ul t s 55 5. 2. 1 C D 40L bl oc ks m i c r ogl i a l pha goc yt os i s of t he A pe pt i de 55 5. 2. 2 C D 40L i nduc t i on o f pr oi n f l a m m a t or y c yt oki ne s by m i c r ogl i a l i s s yne r gi s t i c a l l y e nha nc e d by A 61 5. 2. 3 C D 40 s i gna l l i ng a s s oc i a t e d T h1 but not T h2 c yt ok i ne s i nhi bi t m i c r ogl i a l pha goc yt os i s of A 61 5. 2. 4 C D 40L e nha nc e s M H C I I c o l oc a l i z a t i on w i t h A i n t he m i c r og l i a a s de m ons t r a t e d by c o i m m unobl o t t i ng a nd i m m unof l uor e s c e nc e 66 5. 2. 5 C D 40L i nduc t i on o f M H C I I A pe pt i de c om pl e xe s on m i c r ogl i a i s f unc t i ona l 71 5. 3 D i s c us s i on 75 R E F E R E N C E S 78 A P P E N D I C E S 101 A P P E N D I X I : P ubl i c a t i ons r e l a t i ng t o t hi s di s s e r t a t i on 102 A B O U T T H E A U T H O R E nd pa ge\ni v L I S T O F T A B L E S T a bl e 3. 1 F D A a ppr ove d t r e a t m e nt s f or A l z he i m e r s di s e a s e 5\nv L I S T O F F I G U R E S F i gur e 1 F A C S a na l ys i s of C D 45 i s of or m s on m i c r ogl i a 27 F i gur e 2 C D 45 i s of or m R B a br oga t e s L P S i nduc e d T N F pr oduc t i on by m i c r ogl i a i n v i t r o 29 F i gur e 3 B r a i n T N F l e ve l s i n r e s pons e t o L P S a r e a t t e nua t e d a f t e r a nt i C D 45 p r e t r e a t m e nt 30 F i gur e 4 R T P C R de m ons t r a t e s t ha t L ova s t a t i n i nhi bi t s m i c r ogl i a l C D 40 m R N A e xpr e s s i on i nduc e d by I F N 36 F i gur e 5 I m m unobl ot a nd F A C S a na l ys i s r e ve a l s t ha t L ova s t a t i n i nhi bi t s m i c r ogl i a l C D 40 pr ot e i n e xpr e s s i on i nduc e d by I F N 38 F i gur e 6 L ova s t a t i n t r e a t m e nt oppos e s I F N i nduc e d phos phor yl a t i on of J A K 1/ 2 i n m i c r o gl i a l c e l l s 39 F i gur e 7 L ova s t a t i n t r e a t m e nt oppos e s I F N i nduc e d phos phor yl a t i on of S T A T 1 i n m i c r ogl i a l c e l l s 41 F i gur e 8 L ova s t a t i n oppos e s T N F pr oduc t i on i n m i c r ogl i a l c e l l s i nduc e d by e i t he r I F N / C D 40 o r A # / C D 4 0 c ha l l e nge 43 F i gur e 9 L ova s t a t i n oppos e s I L 1 # pr oduc t i on i n m i c r ogl i a l c e l l s i nduc e d by I F N / C D 40 or A # / C D 40 c ha l l e nge 44 F i gur e 10 L ova s t a t i n oppos e s I L 6 p r oduc t i on i n m i c r og l i a l c e l l s i nduc e d by I F N / C D 40 o r A # / C D 40 c ha l l e nge 45 F i gur e 11 I m m unof l uor e s c e nc e m i c r os c opy de m ons t r a t e s L o va s t a t i n a t t e nua t i on of C D 40 i m pa i r m e nt o f m i c r ogl i a l pha goc yt os i s of A # 46 F i gur e 12 I m m unobl ot a na l ys i s of c e l l a s s oc i a t e d A # de m ons t r a t e s t ha t L ova s t a t i n t r e a t m e nt a t t e nua t e s C D 40 i m pa i r m e nt of\nvi i M i c r ogl i a A c t i va t i on I n A l z he i m e r s D i s e a s e K i r k P hi l l i p T o w ns e nd A B S T R A C T T he w or k de t a i l e d i n t hi s di s s e r t a t i on ha s a n ove r a r c hi ng t he m e of m odul a t i ng m i c r ogl i a a c t i va t i on i n bot h i n v i v o a nd i n v i t r o m o de l s r e l e va nt t o A D T he pr e m i s e i s t ha t unde r s t a ndi ng m i c r ogl i a f unc t i on i n t hi s c ont e xt m a y l e a d t o a b e t t e r unde r s t a ndi ng of A D pa t hoge ne s i s a nd t hus t o e f f e c t i ve t he r a pe ut i c i nt e r ve nt i ons I n c ha pt e r 3 w e e m pl oy a w e l l de f i ne d m ode l o f m i c r og l i a a c t i va t i on w he r e by t he i n t r a pe r i t one a l de l i ve r y of L P S r e s ul t s i n C N S m i c r og l i a a c t i va t i on a nd T N F p r oduc t i on. H a vi ng p r e vi ous l y i de nt i f i e d t ha t C D 45 s i gna l i ng pa t hw a ys a nt a goni z e d m i c r ogl i a T N F pr oduc t i on i n v i t r o a nd gi ve n t ha t i m m unot he r a py w i t h a nt i C D 4 5 a nt i bodi e s a r e a l r e a dy i n c l i ni c a l t r i a l s f or bot h t he t r e a t m e nt of m a l i gna nt di s or de r s a s w e l l a s f or t o l e r a nc e i nduc t i on f ol l ow i ng or ga n t r a ns pl a nt s w e i nve s t i ga t e d w he t h e r m i c r ogl i a l C D 45 c oul d be a r e l e va nt m ol e c ul a r t a r ge t i n t he oppos i t i on o f m i c r ogl i a a c t i va t i on i n v i v o G i ve n t ha t a num be r of e pi de m i ol ogi c a l s t udi e s ha ve s how n a n i nve r s e c or r e l a t i on be t w e e n t he us e of s t a t i ns ( a c l a s s of dr ugs t ha t w e r e i ni t i a l l y de s c r i be d a s s pe c i f i c i nhi bi t or s of c hol e s t e r ol bi os ynt he s i s ) a nd t he i nc i de nc e of A l z he i m e r s di s e a s e ( W ol oz i n, 2000; S i m on 2002; Z a m r i ni 2004 ) ; i n c ha pt e r 4, w e e m pl oye d one of t he m os t pr e s c r i be d s t a t i ns l ova s t a t i n, i n our m i c r ogl i a a c t i va t i on pa r a di gm I nt e r e s t i ngl y w e\nvi i i s how t ha t s t a t i ns not onl y i nhi bi t t he e nz ym e H M G C oA ( t he r a t e l i m i t i ng s t e p of c hol e s t e r ol bi os ynt he s i s ) but t ha t t he y a l s o di s pl a y pl e i ot r ophi c e f f e c t s i nc l udi ng t he i nhi bi t i on of C D 40 e xpr e s s i on. G i ve n t he r ol e o f C D 40 i n m i c r ogl i a a c t i va t i on a nd i t s pot e nt i a l r ol e i n A D pa t hoge ne s i s w e i nve s t i ga t e d w he t he r l ova s t a t i n s pr ot e c t i on i n A D m i ght be de r i ve d f r o m e f f e c t s on m i c r ogl i a f unc t i o n a s gove r ne d by t he C D 40 pa t hw a y. I n c ha pt e r 5 w e p r ovi de e vi de nc e f o r a m ode l o f m i c r ogl i a a c t i va t i on t ha t a ddr e s s e s s om e of t he c ur r e nt c ont r ove r s i e s c onc e r ni ng t he r ol e of m i c r ogl i a c e l l s i n A D pa t hoge ne s i s T he m ode l s ugge s t s t ha t m i c r ogl i a c e l l s e xi s t i n a num be r of di s t i nc t a c t i va t i on s t a t e s ( one s uc h s t a t e s w e de not e t he pha goc yt i c s t a t e ) m i c r ogl i a f unc t i on t o c l e a r c e l l ul a r de br i s o r f or e i gn i nva de r s or i n t he c a s e of A D t o r e m ove a m yl oi d / A pe pt i de s H ow e ve r i n r e s pons e t o c e r t a i n c o s t i m ul i ( i e C D 40 a c t i va t i on) t he s e m i c r ogl i a t a ke t he f or m of a n a nt i ge n p r e s e nt i ng c e l l ( A P C ) w he r e by t he y l os e t he i r pha goc yt i c c a pa c i t y i n l i e u o f c yt oki ne pr oduc t i on a nd t hus pot e nt i a l l y c ont r i but e t o A D pa t hoge ne s i s\n1 C H A P T E R O N E I N T R O D U C T I O N 1. 1 E p i d e m i ol ogy of A l z h e i m e r s d i s e as e O r i gi na l l y de s c r i be d by A l o i s A l z he i m e r i n 1906, A l z he i m e r 's di s e a s e ( A D ) ha s e m e r ge d a s t he m os t c om m on t ype of de m e nt i a i n t he e l de r l y t oda y. A l t hough t he de f i ni t i ve di a gnos i s of A D r e qui r e s hi s t ol ogi c a l c o nf i r m a t i on, t he c l i n i c i a n m a y e s t a bl i s h t he di a gnos i s a nt e m or t e m w i t h a f a i r de g r e e of c e r t a i nt y, ba s e d on t he c l i ni c a l f i ndi ngs of a gr a dua l l y pr og r e s s i ve c ogni t i ve de c l i ne t ha t r e s ul t s i n t he l os s of m e m or y l a ngua ge s ki l l s a nd a c t i vi t i e s of da i l y l i vi ng. A s of 2003, a ppr oxi m a t e l y 4. 5 m i l l i on pe opl e i n t h e U ni t e d S t a t e s w e r e c l i ni c a l l y di a gnos e d w i t h A D a nd t hi s f i gur e i s e xpe c t e d t o t r i pl e by t he ye a r 2050 a c c or di ng t o t he N a t i ona l I ns t i t ut e s of H e a l t h T hi s p r e va l e nc e i s due i n pa r t t o t he f a c t t ha t a gi ng p r e s e nt s a s t r ong r i s k f a c t or f or de ve l opi ng A D f o r e xa m pl e a t a ge 60 t he l i ke l i hood o f de ve l opi ng A D i s e s t i m a t e d t o be 1% ; how e ve r t h e r i s k doubl e s e ve r y f i ve ye a r s r e a c hi ng 30 50% by t he a ge of 85. I nt e r e s t i ngl y, e ve n a f t e r s t a t i s t i c a l c or r e c t i on f or l onge vi t y, w om e n a r e a f f e c t e d w i t h A D a t a r a t i o o f 2: 1 c om pa r e d t o m e n ( C or de r 2004 ) R e c e nt r e s e a r c h s ugge s t s t ha t t he hor m one e s t r oge n c a n be ne ur opr o t e c t i ve a nd i t s r e duc t i on i n pos t m e nopa us a l w om e n m i ght i nc r e a s e t he i r r i s k o f de ve l opi ng A D A l t hough A D pa t hoge ne s i s i s m ul t i f a c t or i a l ge ne t i c r i s k f a c t or s a r e c l e a r l y i nvo l ve d. T he t w o ba s i c t ype s of A D a r e f a m i l i a l a nd s por a di c T he f a m i l i a l A D ( F A D ) i s a n e a r l y\n2 ons e t f or m ( m e a ni ng t he d i s e a s e de ve l ops be f or e a ge 65 ) t ha t c om pr i s e s l e s s t ha n 10 % of A D pa t i e nt s T he F A D e t i ol ogy i s c a us e d by ge ne m ut a t i ons on c hr om os om e s 1 ( P r e s e ni l i n 2) 14 ( P r e s e ni l i n 1) a nd 21 ( A m yl oi d P r e c ur s or P r o t e i n) a l l of w hi c h l e a d t o i nc r e a s e d A pe pt i de pr oduc t i on a nd/ or i t s de pos i t i on i n t he C N S T he s por a di c f o r m of A D ( ha vi ng no know n c a us e or obvi ous i nhe r i t a nc e pa t t e r n) c ons t i t ut e s t he m a j or i t y of c a s e s w i t h ons e t us ua l l y de ve l opi ng a f t e r a ge 65. A l t hough a s pe c i f i c ge ne ha s not be e n i de nt i f i e d a s t he c a us e of l a t e ons e t A D ge ne t i c f a c t or s do a ppe a r t o pl a y a r ol e i n t he de ve l opm e nt of t hi s f or m of A D G e ne t i c a s s oc i a t i on a nd l i nka ge s t udi e s ha ve s how n t ha t t he a pol i popr ot e i n E ( A po E ) ge ne f ound on c hr om os om e 19, i s one r i s k f a c t or ge ne i de nt i f i e d w i t h a n i nc r e a s e d r i s k of de ve l opi ng l a t e ons e t A D T hi s ge ne c ode s f o r a pr ot e i n t ha t he l ps c a r r y c hol e s t e r ol i n t he b l oods t r e a m T he A P O E ge ne ha s s e ve r a l a l l e l e s A P O E e 2, e 3 a nd e 4 T he A po E 4 ge not ype i s a s s oc i a t e d w i t h a n i nc r e a s e d r i s k f o r l a t e ons e t A D w hi l e t he A poE 2 s e e m s t o c onf e r p r ot e c t i on. C ons i s t e nt w i t h a m ul t i f a c t or i a l e t i o l ogy, A poE s t a t us a l one doe s not s e r ve a s a s uf f i c i e nt s c r e e ni ng t oo l a s i t c a nnot de f i ni t i ve l y pr e di c t w hi c h c a r r i e r s w i l l de ve l op A D w he n ons e t w i l l oc c ur or t he s e ve r i t y of t he di s e a s e c our s e 1. 2 S i gn an d S ym p t om s W hi l e s hor t t e r m m e m or y i m pa i r m e nt i s of t e n t he pr e s e nt i ng s ym pt om r e m ot e m e m or y l os s a l s o a ppe a r s t o be a f f e c t e d ove r t i m e A not he r i m por t a nt f e a t ur e of A D i s t he di s t ur ba nc e of l a ngua ge I ni t i a l l y A D pa t i e nt s m a y s e a r c h f or w or ds w he n na m i ng obj e c t s or w hi l e e nga ge d i n a s i m pl e c onve r s a t i on. W i t h pr og r e s s i on of t he di s e a s e how e ve r t he l a ngua ge di f f i c ul t i e s e vol ve i nt o a c om m uni c a t i on br e a kdow n a s t he pa t i e nt\n3 s t r uggl e s w i t h a m a r ke dl y l i m i t e d voc a bul a r y, no m i na l a pha s i a e c hol a l i a a nd de f e c t s i n ve r ba l c om pr e he ns i on. B e ha vi or a l pr obl e m s e m e r ge t hr o ughout t he va r i ou s s t a ge s of t he di s e a s e M ood di s t ur ba nc e s s uc h a s de pr e s s i on, a nxi e t y, o r a pa t hy m a y be p r e s e nt e a r l y on i n A D w hi l e de l us i ons ha l l uc i na t i ons a nd ps yc hos i s c a n be pr o m i ne nt i n l a t e r s t a ge s I n a ddi t i on a ggr e s s i on a nd i na ppr opr i a t e s e xua l be ha vi or c a n be pa r t i c ul a r l y p r obl e m a t i c f o r t he c a r e gi ve r I n a dva nc e d s t a ge s of A D pa t i e nt s m a y e xhi bi t e xt r a pyr a m i da l s i gns s uc h a s t r e m or a nd ga i t di s t ur ba nc e f r ont a l l obe r e l e a s e phe nom e na ur i na r y i nc ont i ne nc e a nd m yoc l onus S e i z ur e s c a n a l s o be s e e n i n s om e pa t i e nt s w i t h l a t e s t a ge di s e a s e F i na l l y pa t i e nt s w i t h e nd s t a ge A D a l m os t i nva r i a bl y e nt e r a ve ge t a t i ve l i ke s t a t e a s a l l c ogni t i ve a c t i vi t y c e a s e s 1. 3 D i agn os i s I n 1984 t he N a t i ona l I ns t i t ut e of N e ur ol ogi c a l a nd C om m uni c a t i ve D i s or de r s a nd S t r oke a nd t he A l z he i m e r 's D i s e a s e a nd R e l a t e d D i s e a s e s A s s o c i a t i on ( N I N C D S A R D A ) e s t a bl i s he d di a gnos t i c c r i t e r i a de s i gne d f or r e s e a r c h pur pos e s a nd c l i ni c a l de f i ni t i on ( M c K ha n n, 1984) N ow r e c om m e nde d by t he A m e r i c a n A c a de m y of N e ur ol ogy ( A A N ) f or t he di a gnos i s of A D t hi s c l a s s i f i c a t i on i s di vi d e d i nt o de f i ni t e p r oba bl e a nd pos s i bl e A D ( K nopm a n 2001) I n a ddi t i on t o hi s t opa t hol ogi c a l c onf i r m a t i on de f i ni t e A D r e qui r e s t h e c l i ni c a l f i ndi ng of de m e nt i a a s de t e r m i ne d by t he M i ni M e nt a l S t a t us E xa m ( M M S E ) or ot he r\n4 s t a nda r di z e d ne ur ops yc hol ogi c a l t e s t i ng; t he e xa m m us t de m ons t r a t e de f i c i t s i n t w o or m or e a r e a s of c ogni t i on w i t h pr ogr e s s i ve m e m or y l os s i n t he a bs e nc e of de l i r i u m P r oba bl e A D i s t he c l i ni c a l de t e r m i na t i on of de m e nt i a a s de s c r i be d a bove a nd i s s uppor t e d by t he f i ndi ngs of i m pa i r e d a c t i vi t i e s of da i l y l i vi ng, l os s of s pe c i f i c c ogni t i ve f unc t i ons s uc h a s l a ngua ge a nd m ot or s ki l l s a nd a f a m i l y hi s t or y O t he r c l i ni c a l f i ndi ngs t ha t s uppor t t he d i a gnos i s i n pa t i e nt s w i t h a dva nc e d A D a r e m yoc l onus ga i t d i s or de r a nd i nc r e a s e d m us c l e t one P os s i bl e A D i s c ons i de r e d w he n t he r e i s va r i a t i on i n t he ons e t pr e s e nt a t i on, or c l i ni c a l c our s e of t he de m e nt i a a nd w he n a s e c ond br a i n di s e a s e or s ys t e m i c di s or de r i s pr e s e nt C l i ni c a l f a c t or s t ha t m a ke t he di a gnos i s of A D un l i ke l y i nc l ude s udde n ons e t a nd f oc a l ne ur ol ogi c a l f i ndi n gs s uc h a s he m i pa r e s i s a nd vi s ua l f i e l d de f i c i t s C l i ni c a l l y bot h t he N I N C D S A D R D A c r i t e r i a a nd t he D i agnos t i c and St at i s t i c al M anual of M e nt a l D i s or de r s I I I R de f i ni t i on of D e m e nt i a of t he A l z he i m e r T ype ( D A T ) m a y be us e d t o d i a gnos e A D w i t h 90 % a c c ur a c y ( K nopm a n, 2001) 1. 4 T r e at m e n t 1. 4. 1 M an age m e n t of C og n i t i on C ur r e nt l y t he r e i s no c ur e f or A D a va i l a bl e t r e a t m e nt s onl y t r a ns i e nt l y i m p r ove m e m or y a nd c ogni t i on A s of 2004, t he r e a r e onl y f i ve F D A a ppr ove d dr ugs c ur r e nt l y a va i l a bl e f or us e i n A D t he r a py a s out l i ne d i n T a bl e I I n pa t i e nt s w i t h m i l d t o m ode r a t e di s e a s e t he m a i ns t a ys of t r e a t m e nt a r e t he a c e t yl c hol i ne s t e r a s e i nhi bi t or s w hi c h a r e a i m e d a t i nc r e a s i ng a c e t yl c hol i ne l e ve l s t hr oughou t t he C N S\n6 I n a ddi t i on t o t he us e of c hol i ne s t e r a s e i nhi bi t or s vi t a m i n E a nd s e l e gi l i ne m a y a l s o be of be ne f i t i n t he pha r m a c ol ogi c t r e a t m e nt o f A D ( D oody 2001 ) T he s e a nt i oxi da nt a ge nt s a r e t hought t o p r ovi de s om e de g r e e of ne ur op r ot e c t i on a nd i n a c l i ni c a l s t udy s l ow e d di s e a s e pr ogr e s s i on by 25% ove r p l a c e bo ( S a no, 1997) H ow e ve r c l i ni c a l j udgm e nt ne e ds t o be c a r e f ul l y e xe r c i s e d w he n pr e s c r i bi ng s e l e gi l i ne f or A D pa t i e nt s a s t he s i de e f f e c t pr o f i l e o f t he m ono a m i ne oxi da s e i nhi bi t or m a y out w e i gh t he be ne f i t o f i t s us e E s t r oge n, t he a c e t yl c hol i ne p r e c ur s or l e c i t hi n, ni c ot i ne a nd m us c a r i ni c a goni s t s ha ve not s how n a ny s i gni f i c a nt be ne f i t i n pa s t c l i ni c a l t r i a l s a nd a r e not a ppr ove d f or t r e a t m e nt 1. 4. 2 M an age m e n t of N on c ogn i t i ve S y m p t o m s D e pr e s s i on i s c om m on i n pa t i e nt s w i t h A D a nd m a y r e qui r e pha r m a c ol ogi c t r e a t m e nt S e r ot oni n r e upt a ke i nhi b i t or s a r e r e l a t i v e l y w e l l t ol e r a t e d by pa t i e nt s a nd a r e pr e f e r r e d ove r t r i c yc l i c a nt i de pr e s s a nt s w hi c h c a n of t e n e xa c e r ba t e t he c ogni t i ve i m pa i r m e nt due t o i t s a nt i c hol i ne r g i c pr ope r t i e s T he oc c ur r e nc e of be ha vi o r a l di s t ur ba nc e s s uc h a s ps yc ho s i s a nd a gi t a t i on r e qui r e s a n i nve s t i ga t i on f or a c or r e c t a bl e unde r l yi ng c a us e s uc h a s a ur i na r y t r a c t i n f e c t i on b e f or e a ne ur ol e pt i c a ge nt s houl d be c ons i de r e d. I f t he r e i s no e xt e r na l e t i ol ogy t he n t h e e s t a bl i s hm e nt of a qui e t c ont r ol l e d, a nd f a m i l i a r e nvi r onm e nt f or t he pa t i e nt c a n he l p t o de c r e a s e c onf us i on a nd di s or i e nt a t i on. S pe c i a l c a r e uni t s w i t hi n l ong t e r m c a r e f a c i l i t i e s m a y be c ons i de r e d a s s om e s t udi e s ha ve s how n a r e duc e d ne e d f or a nt i ps yc hot i c s a nd phys i c a l r e s t r a i nt s a s w e l l a s a de c r e a s e i n be ha vi or a l di s t ur ba nc e s i n A D pa t i e nt s w ho r e s i de t he r e ( D oody, 2001)\n7 F i na l l y, ps yc hos oc i a l i nt e r ve nt i on f or t he c a r e gi ve r i s a n i nt e gr a l pa r t of m a na gi ng pa t i e nt s w i t h A D E duc a t i on s uppor t gr oups a nd r e s pi t e c a r e s e r vi c e s a r e i nva l ua bl e t o f a m i l y m e m be r s a nd f r i e nds w ho pr ovi de t he p r i m a r y c a r e f o r A D pa t i e nt s ( H a l e y, 1997 ) 1. 5 P at h op h ys i ol ogy of A l z h e i m e r s d i s e as e A l t hough i m a gi ng t e c hnol ogi e s a r e r a pi dl y de ve l opi ng t o de t e r m i ne t he e xt e nt of a m yl oi d pl a que bur de n i n t he l i vi ng pa t i e nt w i t h t he i nt e nt of di a gnos i ng, m oni t or i ng t r e a t m e nt e f f e c t i ve ne s s a nd/ or f ol l ow i ng di s e a s e pr ogr e s s i on, c ur r e nt c r i t e r i a l i m i t s a de f i ni t e di a gnos i s of A D t o pos t m or t e m br a i n e xa m i na t i on. T he ha l l m a r k ne ur opa t hol ogi c a l f i ndi ngs i n t he A D br a i n i nc l ude : a m yl oi d pl a que s ; ne ur o f i br i l l a r y t a ngl e s ; a nd ne ur ona l c e l l de a t h A ddi t i ona l pa t hol ogi c a l f i ndi ngs i nc l ude C N S i nf l a m m a t i on a nd g l i os i s ( bot h m i c r ogl i a l a nd a s t r oc yt i c ) 1. 5. 1 A m yl oi d P l a q u e s a m yl oi d pl a que s a r e e xt r a c e l l ul a r pr ot e i na e ous a ggr e gr a t e s pr i nc i pl y f ound i n t he hi ppoc a m pus a nd f r ont a l c or t e x o f A D s uf f e r e r s T he s e a m yl oi d pl a que s or s e ni l e pl aque s m a y be c l a s s i f i e d f ur t he r a c c or di ng t o t he i r s i z e de ns i t y a nd c om pos i t i on, but t he c om m on f e a t ur e i s t ha t t he y a l l c ont a i n i s of o r m s of t he a m yl oi d pr ot e i n ( A ) A i s a 39 42 a m i no a c i d pe pt i de t ha t i s f or m e d by t he pr o t e ol yt i c c l e a va ge of i t s pa r e nt m ol e c ul e a m yl oi d pr e c ur s or p r ot e i n ( A P P ) by e nz ym e s r e f e r r e d t o a s s e c r e t a s e s S e ve r a l l i ne s of e vi de nc e s ugge s t t ha t A de pos i t i on i n t he C N S m a y be c e nt r a l t o t he pa t hoge ne s i s of A D F or i ns t a nc e s pe c i f i c m ut a t i ons i n t he A P P g e ne a s w e l l a s i t s s e c r e t a s e s ha ve be e n i de nt i f i e d i n pa t i e nt s w i t h t he f a m i l i a l f or m of A D ( M or a n, 1995) F u r t he r m or e t he A P P\n8 ge ne r e s i de s on c hr om os om e 21 a nd t he i nc r e a s e i n ge ne dos a ge s e e n i n t he t r i s om y di s or de r of D ow n's s yndr om e ha s be e n c or r e l a t e d w i t h ove r e xpr e s s i on of t he A P P m R N A i n t he D ow n s s yndr om e br a i ns ( L e ndon, 1 997) I m po r t a nt l y, i ndi vi dua l s w i t h D ow n s yndr om e ( by 40 ye a r s of a ge ) de ve l op vi r t u a l l y a l l of t he m o r phol ogi c a l ne ur od e ge ne r a t i ve a nd bi oc he m i c a l c ha nge s obs e r ve d i n A D b r a i ns i nc l udi ng a m yl oi d pl a que s a nd ne ur of i br i l l a r y t a ngl e s ( H ow a r d 1995 ) T he s e f i ndi ngs f o r m t he ba s i s of t he a m yl oi d hypo t he s i s of A D pa t hoge ne s i s w hi c h s t a t e s t ha t t he e l e va t e d C N S l e ve l s of A a nd i t s de ve l opm e nt i nt o a m yl o i d pl a que s l e a ds t o ne ur ode ge ne r a t i on a s e vi de nc e d by ne ur of i br i l l a r y t a ngl e f o r m a t i on. A l t hough t he a m yl oi d hypot he s i s i s a r gua bl y t he m os t pr e va l e nt e xpl a na t i on f o r A D di s e a s e i t i s no t w i t hout i t s w e a kne s s e s e vi de nc e d pr i m a r i l y by t he poo r c or r e l a t i on be t w e e n t he c onc e nt r a t i ons a nd di s t r i but i on of a m y l oi d de pos i t i ons i n t he br a i n a nd s e ve r a l pa r a m e t e r s of A D pa t hol ogy, i nc l ud i ng de gr e e o f de m e nt i a l os s of s yna ps e s l os s of ne ur ons a nd a bnor m a l i t i e s of t he c yt os ke l e t o n. F ur t he r m or e a l t hough t r a ns ge ni c m ous e m ode l s t h a t ha r bor f a m i l i a l A D f or m s of hu m a n A P P a nd/ or P S 1 do di s pl a y c opi ous a m ount s of a m yl oi d pl a que s i n t he i r br a i ns t he r e i s no obvi ous e f f e c t on ne ur on f unc t i on o r vi a bi l i t y s ugge s t i ng t ha t a l t hough A pe pt i de s m a y be t he t r i gge r f or t he ne ur ode ge ne r a t i ve e ve nt s s e e n i n t he A D br a i n t he dow ns t r e a m ne ur ot oxi c e f f e c t or f unc t i on l i e s e l s e w he r e I nt e r e s t i ngl y, a ni m a l m ode l s t ha t e xpr e s s hum a n t a u pr ot e i n do i n f a c t m a ni f e s t s i gni f i c a nt ne ur on c e l l l os s i n t he a bs e nc e of a m yl oi d de pos i t s ( Z ha ng 2004) 1. 5. 2 N e u r of i b r i l l ar y T an gl e s\n9 N e ur of i br i l l a r y t a ngl e s e xa m i ne d a t t he e l e c t r on m i c r os c opy l e ve l r e ve a l a n a r r a y of pa i r e d he l i c a l f i l a m e nt s w hi c h b i oc he m i c a l a na l ys i s ha s s how n t o be c om pos e d pr i nc i pl y of t he p r ot e i n t a u T a u i s a n i m por t a nt c y t os ke l e t a l pr ot e i n, w hi c h i n t he ne ur on i s e s s e nt i a l f or a xona l gr ow t h a nd r e m ode l i ng H o w e ve r w he n hype r phos phor yl a t e d t he t a u pr ot e i n f or m s t a ngl e s t ha t a r e s ys t e m a t i c a l l y de pos i t e d w i t hi n ne ur ons l oc a t e d i n t he hi ppoc a m pus a nd m e di a l t e m por a l l obe t he pa r i e t o t e m por a l r e gi on, a nd t he f r ont a l a s s oc i a t i on c or t i c e s ul t i m a t e l y l e a di ng t o c e l l de a t h. A put a t i ve l i nk be t w e e n a m yl oi d pl a que de pos i t i on a nd ne ur of i b r i l l a r y t a ngl e f o r m a t i on i s a c e nt r a l poi nt o f t he a m yl oi d hypot he s i s ye t f i ndi ng t he c e l l ul a r a nd/ or m ol e c ul a r s i gna l i ng pa t hw a ys t ha t br i dge s t he s e t w o pa t hol ogi c a l f e a t ur e s r e m a i ns a n a r e a of i nt e ns e r e s e a r c h. I nt e r e s t i ngl y, C N S a r e a s i nvol ve d w i t h a m yl oi d pl a que s a r e not s t r i c t l y m i r r o r e d by ne ur o f i b r i l l a r y t a ngl e f or m a t i on, but a r e a s of ne ur ona l c e l l de a t h a nd s yna ps e l os s ha ve a s i m i l a r di s t r i but i on pa t t e r n a s t he ne ur of i br i l l a r y t a ngl e s 1. 5. 3 I n f l am m at i o n an d G l i os i s T he e xa c t r ol e of i nf l a m m a t i on i n t he pa t hoge ne s i s of A D i s s t i l l c ont r ove r s i a l A t t he c e l l ul a r l e ve l t hi s i nf l a m m a t i on i s e vi de nc e d ( upon pos t m or t e m e xa m i na t i on ) by t he pr e s e nc e of m i c r ogl i a a nd a s t r oc yt e s a r ound t h e a m yl oi d pl a que s ( A l z he i m e r 1907; A ki ya m a 1994) T hi s ha s be e n r e c e nt l y i nt e r p r e t e d t o r e p r e s e nt m i c r ogl i a pha goc yt i c c l e a r a nc e or a s t r oc yt e s w a l l i ng of f of t he a m yl oi d pl a que s B e s i de s t he i r pha goc yt i c r ol e m i c r ogl i a c a n a l s o pr oduc e p r o i nf l a m m a t or y m ol e c ul e s w hi c h i s pa r t of t he a r m a m e nt a r i um t o de a l w i t h i nva di ng or ga ni s m s T hi s dua l r o l e of t he a c t i va t e d m i c r ogl i a ha s l e d t o t he va r i ous hypot he s e s r e ga r di ng t he i r i nvol ve m e nt i n A D O n one\n10 ha nd, t he m i c r ogl i a dys f unc t i on hypot he s i s s ugge s t s t ha t t he s e ne s c e nc e of m i c r ogl i a l e a ds t o a c c um ul a t i on of a m yl o i d pl a que s due t o t he l os s of t he i r pow e r f u l c l e a r a nc e m e c ha ni s m s O n t he ot he r ha nd, c hr oni c m i c r ogl i a a c t i va t i on l e a di ng t o e l e va t e d pr oi nf l a m m a t or y pr oduc t s ha s be e n s how n t o m e di a t e ne ur ot oxi c i t y i n v i t r o A t t he m ol e c ul a r l e ve l di f f e r e nt i a l di s pl a y, ge ne a r r a y, a s w e l l a s pr ot e i n s t udi e s ha ve s ugge s t e d t ha t va r i ous pr o i nf l a m m a t or y m ol e c ul e s a r e e l e va t e d i n t he A D br a i n c om pa r e d t o a ge m a t c he d c ogni t i ve nor m a l c ont r ol s M or e ove r e pi de m i ol ogi c a l s t udi e s ha ve s how n t ha t t he us e of non s t e r oi da l a nt i i n f l a m m a t or y dr ugs ( N S A I D S ) a r e a s s oc i a t e d w i t h a de c r e a s e d r i s k f or de ve l opi ng A D H ow e ve r a nu m be r of pr os pe c t i ve c l i ni c a l t r i a l s e va l ua t i ng t he us e of d r ugs t a r ge t i ng va r i ous a s pe c t s of t he i m m une s ys t e m s uc h a s pr e dni s one hydr oxyc hol or oqui ne a nd s e l e c t i ve C O X 2 i nhi bi t or s ha ve onl y be e n a b l e t o de m ons t r a t e m a r gi na l be ne f i t s ( J one s 2001) R e c e nt l y, i t ha s be e n r e por t e d t ha t t he N S A I D S i nhi bi t s e c r e t a s e a c t i vi t y a nd t he r e by A pr oduc t i on ( B e he r 2004) s ugge s t i ng t he pos s i bi l i t y of d i r e c t e f f e c t of N S A I D S on A P P m e t a bol i s m i nde pe nde nt of t he m odul a t i on of t he C N S i n f l a m m a t or y m i l i e u.\n11 C H A P T E R T WO M A T E R I A L S A N D M E T H O D S 2. 1 P e p t i d e s an d d r u gs 2. 1. 1 C y3 A p e p t i d e p r e p ar at i on T he C y3 A pe pt i de s c onj uga t i on w a s c a r r i e d out w i t h s t r i c t a c c or da nc e t o m a nuf a c t ur e r s de s c r i be d pr ot oc ol s B r i e f l y A 1 4 2 w a s di s s ol ve d i n 0. 15 M s odi um c hl or i de a nd C y3 T M m ono r e a c t i ve N H S e s t e r ( A m e r s ha m B i os c i e nc e s P i s c a t a w a y, N J ) w a s di l ut e d i n di m e t hyl s ul f oxi de ( D M S O ) t o a w o r ki ng c onc e nt r a t i on o f 10 m g/ m l a nd t hi s w a s s l ow l y a dde d t o t he A 1 4 2 s ol ut i on w hi l e s t i r r i ng. T he C y3 A 1 4 2 s ol ut i on w a s pr ot e c t e d f r om l i ght w hi l e i t w a s s t i r r e d f or 45 m i n a t r oom t e m pe r a t ur e T o s e pa r a t e t he f r e e C y3 dye t he s ol ut i on w a s di a l yz e d a ga i ns t 1 L of 0 15 M s odi um c hl or i de f or 4 h a t r oom t e m pe r a t ur e T he s ol ut i on w a s t he n e xc ha nge d w i t h f r e s h 0. 15 M s odi um c hl or i de a nd di a l yz e d ove r ni ght a t 4 C T he ne xt da y t he C y3 A 1 4 2 s ol ut i on w a s di a l yz e d a ga i ns t 1L of 0. 1 M P B S f or 4 h a t r oom t e m pe r a t u r e a nd a ga i n di a l yz e d ove r ni ght us i ng f r e s h 0. 1 M P B S T he s ol ut i on w a s t he n f i l t e r e d t h r ough a 0 22 m s yr i nge a nd t he e l ua t e w a s a l i quot e d a nd s t or e d a t 20 C unt i l us e 2. 1. 2 A n t i b od i e s an d r e c om b i n an t p r o t e i n s\n12 L ova s t a t i n w a s obt a i ne d f r om S i gm a ( S t L oui s M O ) a nd di s s ol ve d i n D M S O ( 1% ) t o a s t oc k c onc e nt r a t i on of 25 m M M ous e a nt i hum a n A m onoc l ona l a nt i body, c l one B A M 10, w a s pur c ha s e d f r om S i gm a ( S t L oui s M O ) A 1 4 2 a nd F I T C c onj uga t e d A 1 4 2 pe pt i de s w e r e ob t a i ne d f r o m Q C B / B i o s our c e I nt e r na t i ona l ( C a m a r i l l o C A ) H um a n s ol ubl e r e c om bi na nt C D 40L p r ot e i n w a s pur c ha s e d f r om A l e xi s B i oc he m i c a l s ( S a n D i e go, C A ) R e c om bi na nt p r ot e i ns ( I F N T N F # I L 4 a nd I L 10) w e r e pur c ha s e d f r om R & D S ys t e m s P ur i f i e d r a t a nt i m ous e M H C c l a s s I I a nt i body w a s obt a i ne d f r o m P ha r M i nge n ( S a n D i e go C A ) T he a nt i m ous e a nd a nt i r a bbi t H R P c onj uga t e d s e c ond a nt i bodi e s w e r e pur c ha s e d f r om N e w E ngl a nd B i ol a bs ( B e ve r l y, M A ) I m m un B l ot s pol yvi nyl i de ne di f l uo r i de m e m br a ne s a nd t he I m m un S t a r c he m i l um i ne s c e nc e s ubs t r a t e w e r e pur c ha s e d f r om B i o R a d ( H e r c ul e s C A ) 2. 2 P r i m ar y m u r i n e m i c r ogl i a c e l l c u l t u r e s B r e e di ng pa i r s of C 57B L / 6 m i c e w e r e pur c ha s e d f r om J a c ks on L a bor a t or y ( B a r H a r bo r M E ) a nd hous e d i n t he a ni m a l f a c i l i t y a t t he U ni ve r s i t y of S out h F l or i da H e a l t h S c i e nc e C e nt e r M ur i ne pr i m a r y c ul t ur e m i c r ogl i a l c e l l s w e r e i s ol a t e d f r om m ous e c e r e br a l c or t i c e s a nd w e r e gr ow n i n R P M I m e di um s uppl e m e nt e d w i t h 5% f e t a l c a l f s e r um 2 m M gl ut a m i ne 100 uni t s / m L pe ni c i l l i n, 0. 1 m g/ m L s t r e pt om yc i n, a nd 0 05 m M 2 m e r c a pt oe t ha nol a c c or di ng t o p r e vi ous l y de s c r i be d m e t hods ( T a n 1999 ) B r i e f l y c e r e br a l c or t i c e s f r om ne w bor n m i c e ( 1 2 da ys ol d ) w e r e i s ol a t e d unde r s t e r i l e c ondi t i ons a nd w e r e ke pt a t 4 C p r i or t o m e c ha ni c a l di s s oc i a t i on. C e l l s w e r e pl a t e d i n 75 c m 2 f l a s ks a nd c om pl e t e m e di um w a s a dde d. P r i m a r y c ul t ur e s w e r e ke pt f o r 14 da ys s o t ha t onl y gl i a l c e l l s r e m a i ne d, a nd m i c r ogl i a l c e l l s w e r e i s ol a t e d by s ha ki ng f l a s ks a t 200 r pm\n13 i n a L a b L i ne I nc uba t or S ha ke r M or e t ha n 98 % of t he s e gl i a l c e l l s s t a i ne d pos i t i ve f o r M A C 1 ( C D 11b/ C D 18; B oe hr i nge r M a nnhe i m I n di a na pol i s I N ) A ddi t i ona l l y, be t w e e n 85% a nd 95 % of m i c r ogl i a l c e l l s s t a i ne d pos i t i ve f or C D 45 by f l uor e s c e nc e a c t i va t e d c e l l s or t e r ( F A C S ) a na l ys i s a s de s c r i be d pr e vi ous l y ( T a n, 1999) 2. 3 M i c r ogl i al p h agoc yt os i s as s ay 2. 3. 1 U s i n g f l u or o m e t r i c d e t e c t i o n o f F I T C A F or f l u or om e t i c a na l ys i s p r i m a r y m ur i ne m i c r ogl i a l c e l l s w e r e s e e de d a t 1 x 10 5 c e l l s / w e l l ( n = 6 f or e a c h c ondi t i on) i n 24 w e l l t i s s ue c ul t ur e pl a t e s c ont a i ni ng 0 5 m L o f c om pl e t e m e di um ( R P M I 1640 m e di um s uppl e m e nt e d w i t h 5% f e t a l c a l f s e r um ) T he s e c e l l s w e r e t r e a t e d f or 15, 30 a nd 60 m i n w i t h a ge d A 1 4 2 c onj uga t e d w i t h F I T C ( s uppl i e d by B i o s our c e I nt e r na t i ona l us e d a t 300 n M di s s ol ve d i n dH 2 0 a nd p r e i nc uba t e d f or 24 h a t 37 o C ) ( C hung, 1999) I n t he pr e s e nc e of F I T C A 1 4 2 m i c r ogl i a l c e l l s w e r e t he n c o t r e a t e d w i t h C D 40L or c ont r ol ( he a t i na c t i va t e d C D 40L pr ot e i n 2 g/ m L ) S om e o f t he s e c e l l s w e r e t r e a t e d w i t h a g e d F I T C A 1 4 2 i n t he pr e s e nc e of c yt oki ne s ( i nc l udi ng T N F # I F N I L 4 a nd I L 1 0) f o r 60 m i n. I n a ddi t i on i n pa r a l l e l 24 w e l l t i s s ue c ul t ur e pl a t e s m i c r ogl i a l c e l l s w e r e i nc uba t e d a t 4 o C w i t h F I T C A 1 4 2 w i t h or w i t hout va r i ous c om bi na t i ons of C D 40L or c yt oki ne s M i c r ogl i a l c e l l s w e r e t he n r i ns e d 3 t i m e s i n A f r e e c om pl e t e m e di um a nd t h e m e di a w e r e e xc ha nge d w i t h f r e s h A f r e e c om pl e t e m e di um f or 10 m i n bot h t o a l l o w f or r e m ova l o f non i nc or por a t e d A a nd t o pr om o t e c onc e nt r a t i on o f t he A i nt o pha go s om e s E xt r a c e l l ul a r a nd c e l l a s s oc i a t e d F I T C A w e r e qua nt i f i e d us i ng a M F X 96 w e l l m i c r opl a t e f l uor om e t e r ( M ol e c ul a r D e vi c e s C or p. S unnyva l e C A ) w i t h a n e m i s s i on w a ve l e ngt h of 538 nm a nd\n14 a n e xc i t a t i on w a ve l e ngt h of 485 nm A s t a nda r d c ur ve f r om 0 nM t o 500 nM of F I T C A w a s r un f or e a c h pl a t e T ot a l c e l l ul a r pr ot e i ns w e r e qua nt i f i e d us i ng t he B i o R a d p r ot e i n a s s a y. T he m e a n f l uor e s c e nc e va l ue s f or e a c h s a m pl e a t 37 C a nd 4 C a t t he i ndi c a t e d t i m e poi nt s w e r e de t e r m i ne d by f l uo r om e t i c a na l ys i s R e l a t i ve f ol d c ha nge va l ue s w e r e c a l c ul a t e d a s : ( m e a n f l uor e s c e nc e va l ue f or e a c h s a m pl e a t 37 C / m e a n f l uor e s c e nc e va l ue f or e a c h s a m pl e a t 4 C ) I n t hi s m a nne r bot h e xt r a c e l l ul a r a nd c e l l a s s oc i a t e d F I T C A ! w e r e qua nt i f i e d. ( b ) F or f l uor e s c e nc e m i c r os c ope e xa m i na t i on, pr i m a r y m i c r ogl i a l c e l l s ( s e e de d i n 24 w e l l t i s s ue c ul t ur e pl a t e s ) w e r e t r e a t e d w i t h p r e a ggr e ga t e d P E A 1 4 2 ( 50 nM ) i n t he p r e s e nc e or a bs e nc e of C D 40L ( 2 g/ m L ) a t 37 o C f or 60 m i n. I n a d di t i on i n pa r a l l e l 24 w e l l t i s s ue c ul t ur e pl a t e s m i c r ogl i a l c e l l s w e r e i nc uba t e d a t 4 o C w i t h P E c onj uga t e d A ( 50 nM ) w i t h or w i t hout C D 40L ( 2 g/ m L ) f or 60 m i n F ol l ow i ng t r e a t m e nt t he s e c e l l s w e r e w a s he d 5 t i m e s w i t h i c e c ol d P B S t o r e m ove e xt r a c e l l ul a r A a nd t he n s t a i ne d w i t h F I T C a nt i m ous e M H C I I a nt i body ( 2 g/ m L ; P ha r M i nge n) a t 4 o C f o r 15 m i n A f t e r w a s hi ng 3 t i m e s w i t h i c e c ol d P B S t he s e c e l l s w e r e f i xe d i n 4% pa r a f or m a l de hyde m ount e d a nd t he n vi e w e d unde r a n O l ym pus I X 71/ I X 51 f l uor e s c e nc e m i c r os c ope e qui ppe d w i t h a di gi t a l c a m e r a s ys t e m ( c ) F or W e s t e r n bl ot e xa m i na t i on, pr i m a r y c ul t ur e d m i c r o gl i a l c e l l s w e r e t r e a t e d w i t h C D 40L ( 2 g/ m L ) i n t he pr e s e nc e or a bs e nc e of p r e a ggr e ga t e d A 1 4 2 ( 3 M ) f or 1 h C e l l l ys a t e s w e r e pr e pa r e d a nd i m m unopr e c i pi t a t e d us i ng a nt i m ous e M H C I I a nt i body ( 1: 50; P ha r M i nge n) a nd t he n s ubj e c t e d t o W e s t e r n bl ot u s i ng a nt i hum a n A a nt i body ( B A M 10, 1: 1 500; S i gm a ) ( T a n, 2000) 2. 3. 2 M i c r os c op i c an al ys i s of F l u or e s c e n c e C Y 3 A\n15 M i c r ogl i a l pha goc yt os i s of f i b r i l l a r A pe pt i de s w a s c a r r i e d out s i m i l a r l y t o pr e vi ous l y de s c r i be d pr ot oc ol s ( K i t a m ur a Y 2003; C hung 1 997; W e bs t e r 2001; W ys s C or a y, 2003) M i c r ogl i a l c e l l s w e r e c ul t ur e d i n 6 w e l l t i s s ue c ul t ur e pl a t e s w i t h gl a s s i ns e r t s ( f or f l uor e s c e nc e m i c r os c opy) a t 5 x 1 0 5 / w e l l i n t h e pr e s e nc e or a bs e nc e of C y3 c onj uga t e d A 1 4 2 ( 3 M ) w i t h a goni s t a nt i C D 40 a nt i body ( 2. 5 g/ m L ) or i s ot ype m a t c he d I gG ( 2 5 g / m L ; c ont r ol ) i n t he pr e s e nc e or a bs e nc e of l ova s t a t i n ( 10 M ) I n pa r a l l e l di s he s m i c r ogl i a l c e l l s w e r e i nc uba t e d w i t h C y3 c onj uga t e d A 1 4 2 unde r t he s a m e t r e a t m e nt c ondi t i ons a bove e xc e pt t he y w e r e i nc uba t e d a t 4 o C t o c ont r ol f or non s pe c i f i c a l l y c e l l ul a r a s s oc i a t i on of C y3 A 1 4 2 M i c r ogl i a l c e l l s w e r e t he n r i ns e d 3 t i m e s i n A 1 4 2 f r e e c om pl e t e m e di um a nd t he m e di a w a s e xc ha nge d w i t h f r e s h A 1 4 2 f r e e c om pl e t e m e di um f o r 10 m i n bot h t o a l l ow f o r r e m ova l of non i nc or por a t e d C y3 A 1 4 2 a nd t o pr om o t e c onc e nt r a t i on o f t he C y3 A 1 4 2 pe pt i de i nt o pha gos om e s T hi s m e di um w a s w i t hdr a w n a nd m i c r ogl i a l c e l l w e r e r i ns e d 3 t i m e s w i t h i c e c ol d P B S F o r f l uor e s c e nc e m i c r os c opy, m i c r ogl i a l c e l l s on gl a s s c ove r s l i ps w e r e t he n f i xe d f or 10 m i n a t 4 o C w i t h 4% ( w / v) pa r a f or m a l de hyde ( P F A ) di l ut e d i n P B S A f t e r 3 t i m e s of s uc c e s s i ve r i ns e s i n T r i s buf f e r e d s a l i n e ( T B S ) m i c r ogl i a nuc l e i w e r e de t e c t e d by i nc uba t i on w i t h D A P I f o r 10 m i n. a nd f i na l l y m ou nt e d w i t h f l u or e s c e nc e m ount i ng m e di a c ont a i ni ng S l ow F a de a nt i f a di ng r e a ge nt ( M ol e c ul a r P r obe s E uge ne O R ) a nd t he n vi e w e d unde r a n O l ym pus I X 71/ I X 51 f l uor e s c e nc e m i c r os c ope e qui ppe d w i t h a di gi t a l c a m e r a s ys t e m t o a l l ow f o r di gi t a l c a pt ur e o f i m a g e s 2. 3. 3 I m m u n op r e c i p i t at i on / I m m u n ob l ot an al ys i s of M i c r ogl i al p h agoc yt os i s of A\n16 F or de t e c t i on o f c e l l a s s oc i a t e d A i n t he pr e s e nc e of p r e a ggr e ga t e d A 1 4 2 ( 3 M ) i n pa r a l l e l pr i m a r y m i c r ogl i a l c e l l s w e r e pl a t e d i n 6 w e l l t i s s ue c ul t ur e pl a t e s a t 5 x 10 5 c e l l s / w e l l a nd t r e a t e d w i t h t he s a m e t r e a t e d c ondi t i ons de s c r i be d a bove I m m unobl ot t i ng w a s c a r r i e d out w i t h t he m onoc l ona l a nt i A a nt i b ody ( B A M 10, 1: 1 000 d i l ut i on; S i gm a ) f o l l ow e d by a n a nt i m ous e I gG H R P a s a t r a c e r B l ot s w e r e de ve l ope d us i ng t he I m m un S t a r c he m i l um i ne s c e nc e s ubs t r a t e T he m e m br a ne s w e r e s t r i ppe d a nd t he n r e pr obe d w i t h a r e f e r e nc e a nt i m ous e a c t i n m onoc l o na l a nt i body, w hi c h a l l ow s f or qu a nt i f i c a t i on of t he ba nd de ns i t y r a t i o o f A t o a c t i n by de ns i t om e t r i c a na l ys i s 2. 4 A n al ys i s of c yt o k i n e s b y E L I S A A s de s c r i be d i n pr e vi ous s t udi e s ( T a n 1999) c e l l c ul t ur e m e di a w e r e c ol l e c t e d f or m e a s ur e m e nt of c yt oki ne s by c om m e r c i a l c yt oki ne E L I S A ki t s ( R & D S ys t e m s ) C e l l f r e e s upe r na t a nt s w e r e c ol l e c t e d a nd s t or e d a t 70 C u nt i l a na l ys i s D uoS e t e nz ym e l i nke d i m m unoa bs or ba nc e a s s a y ( E L I S A ) ki t s ( i nc l udi ng T N F # I L 1 I L 6 I L 2 a nd I F N ) w e r e obt a i ne d f r o m R & D S ys t e m s ( M i nne a pol i s M N ) C yt oki ne s l e ve l s i n t he s upe r na t a nt s w e r e e xa m i ne d us i ng t he r e s pe c t i ve c yt oki ne E L I S A i n s t r i c t a c c or da nc e w i t h t he m a nuf a c t ur e r 's pr ot oc ol s C e l l l ys a t e s w e r e a l s o pr e pa r e d a nd t he B i o R a d ( H e r c ul e s C A ) pr o t e i n a s s a y w a s pe r f or m e d t o m e a s ur e t ot a l c e l l ul a r p r ot e i n f r om e a c h of t he c e l l gr oups D a t a a r e r e pr e s e nt e d a s pg/ m g t ot a l c e l l ul a r p r ot e i n f o r e a c h c yt oki ne 2. 5 A n t i ge n p r e s e n t at i on as s ay W e i nt r a pe r i t one a l l y i m m uni z e d C 57B L / 6 m i c e a t 4 w e e ks of a ge w i t h a s i ngl e dos e of A 1 4 2 or ve hi c l e c ont r ol ( phos pha t e buf f e r e d s a l i n e P B S ) e m ul s i f i e d 1: 1 ( v/ v ) i n\n17 c om pl e t e F r e und s a dj uva nt S e ve n t o t e n da ys l a t e r t he s e m i c e w e r e s a c r i f i c e d, t he i r s pl e e ns w e r e i s ol a t e d a nd hom oge ni z e d i ndi vi dua l l y i n 10 m L of c om pl e t e m e di um ( T a n 2 000) c e nt r i f uge d a t 5, 000 x g r e s us pe nde d i n 3 m L of R B C l ys i s ( G e nt r a S ys t e m s M i nne a pol i s M N ) a nd a l l ow e d t o s t a nd on i c e f or 10 m i n. S e ve n m i l l i l i t e r s of c om pl e t e m e di um w a s t he n a dde d t o s pl e e n hom oge na t e s t h e m i xt ur e w a s c e nt r i f uge d a ga i n a t 5, 00 0 x g, t he s upe r na t a nt s w e r e r e m ove d a nd t he r e s ul t i ng s pl e noc yt e s w e r e r e s us pe nde d i n 10 m L of c om pl e t e m e di um C D 4 + T c e l l s w e r e i s ol a t e d f r om t he s e s pl e noc yt e s a s pr e vi ous l y de s c r i be d ( T a n, 2000 ) a n d di s t r i but e d a t a c onc e nt r a t i on o f 1 x 10 6 c e l l s / w e l l i n 24 w e l l pl a t e s a nd c o c ul t ur e d w i t h A 1 4 2 p r e t r e a t e d m i c r ogl i a l c e l l s a t a c onc e nt r a t i on of 1 x 10 5 / w e l l i n 1 m L of m e di a M i c r ogl i a l c e l l s ha d be e n pr e t r e a t e d w i t h A 1 4 2 e i t he r i n t he p r e s e nc e or t he a bs e nc e of C D 40L pr ot e i n f o r 6 h pr i or t o c o c ul t ur e ( T a n, 2002 ) T he r e s ul t i ng c o c ul t u r e d s upe r na t a nt s w e r e c ol l e c t e d f or a na l ys i s of c yt oki ne s I F N I L 2 T N F # a nd I L 6 w e r e m e a s ur e d by E L I S A a s pr e vi ous l y r e por t e d ( T a n, 2000, 2002) T o t a l c e l l ul a r pr ot e i ns of e a c h s a m pl e w e r e qua nt i f i e d us i ng t he B i o R a d pr ot e i n a s s a y, a l l ow i ng r e s ul t s t o be e xpr e s s e d a s pg of e a c h c yt oki ne / m g o f t ot a l c e l l ul a r p r ot e i n. 2. 6 C D 40 d e t e c t i on i n m i c r ogl i al c e l l s 2. 6. 1 C D 40 m R N A d e t e c t i on b y R e ve r s e t r an s c r i p t as e P C R an al ys i s ( R T P C R ) P r i m a r y c ul t u r e d m i c r ogl i a l c e l l s w e r e p l a t e d a t 5 x 10 5 c e l l s / w e l l i n 6 w e l l t i s s ue c ul t ur e pl a t e s ( C os t a r C a m br i dge M A ) a nd s t i m ul a t e d f or 3 h w i t h I F N ( 100 U / m L ) i n t he pr e s e nc e or a bs e nc e of l ova s t a t i n ( 10 M ) T ot a l R N A w a s i s ol a t e d f r om m i c r og l i a c e l l c ul t ur e s us i ng T r i z ol r e a ge nt ( I nvi t r oge n C a r l s ba d C a l i f or ni a ) a s r e c om m e nde d i n t he\n18 m a nuf a c t ur e r 's pr ot oc ol R N A c onc e nt r a t i on w a s m e a s ur e d by s p e c t r ophot om e t r y a t 260 nm R T P C R w a s pe r f or m e d a s de s c r i be d pr e vi ous l y ( T a n, 1999 a ) B r i e f l y c D N A w a s pr e pa r e d by m i xi ng 1 g of t ot a l R N A f r om e a c h t r e a t m e nt w i t h a n ol i go ( dT ) p r i m e r a nd t he M M L V r e ve r s e t r a ns c r i pt a s e ( I nv i t r oge n) ; t he r e a c t i on m i x w a s i nc uba t e d i n a 37 C w a t e r ba t h f o r 50 m i n be f or e he a t i na c t i va t i on of t he m i x by i nc r e a s i ng t he t e m pe r a t ur e t o 70 C f or 10 m i n. T h i s c D N A r e a c t i on m i xt ur e ( 20 L ) w a s di l ut e d w i t h 180 l o f D N A a s e / R N A a s e f r e e w a t e r a nd 10 L of t he c D N A s ol ut i on w a s us e d f or ge ne s pe c i f i c P C R T he P C R pr i m e r s us e d w e r e C D 40 s e ns e : 5 C T G C C C A G T C G G C T T C T T C T C 3 a nd a nt i s e ns e : 5 C C T G T G T G A C A G G C T G A C A C 3 ol i gonuc l e ot i de s w e r e de s i gne d t o pr oduc e t he pa r t i a l 374 bp m ous e C D 40 c D N A [ 37] ( T one , 2000) ; a c t i n s e ns e : 5' C A G A G G C T C C C C T A A A T C C C 3' a nd a c t i n a nt i s e ns e : 5' C A C A C T G A G T A C T T G C G C T C 3', w hi c h yi e l ds t he 702 bp a c t i n c D N A f r a gm e nt A c t i n w a s a m pl i f i e d f r om a l l s a m pl e s a s a hous e ke e pi ng ge ne t o nor m a l i z e e xpr e s s i on. A c ont r ol ( no t e m pl a t e ) w a s i nc l ude d f or e a c h pr i m e r s e t P C R w a s pe r f or m e d w i t h e a c h c yc l e c ons i s t i ng of 94C f or 1 m i n, 55C f o r 2 m i n a nd 72C f or 2 m i n, f ol l ow e d by a f i na l e xt e ns i on s t e p a t 72 C f or 10 m i n P C R c yc l e num be r s w e r e ke pt l ow t o pe r f o r m s e m i qua nt i t a t i ve P C R ( a c t i n, 18 c yc l e s ; C D 40 20 c yc l e s ) P C R pr oduc t s w e r e r e s ol ve d on 1 2% e t hi di um br o m i de s t a i ne d a ga r os e ge l s a nd vi s ua l i z e d by ul t r a vi ol e t t r a ns i l l um i na t i on. 2. 6. 2 M i c r ogl i al c e l l s u r f ac e C D 40 p r ot e i n d e t e c t i on b y f l ow c yt om e t r i c an al ys i s F or f l ow c yt om e t r i c a na l ys i s of m i c r ogl i a l C D 40 e xpr e s s i on, pr i m a r y c ul t ur e d m i c r ogl i a l c e l l s w e r e pl a t e d i n 6 w e l l t i s s ue c ul t ur e pl a t e s a t 5 x 10 5 c e l l s / w e l l a nd i nc uba t e d w i t h\n19 I F N ( 100 U / m L ) i n t he pr e s e nc e or a bs e nc e of l o va s t a t i n ( 10 M ) T w e l ve hour s a f t e r i nc uba t i on, m i c r ogl i a l c e l l s w e r e w a s he d w i t h f l ow buf f e r [ P B S c ont a i ni ng 0 1% ( w / v) s odi um a z i de a nd 2% ( v/ v) F C S ] a nd r e s us pe nde d i n 250 l o f i c e c ol d f l ow buf f e r f o r f l uor e s c e nc e a c t i va t e d c e l l s or t i ng ( F A C S ) a na l ys i s a c c or di ng t o m e t hods de s c r i be d pr e vi ou s l y ( T a n 1999 c ) B r i e f l y c e l l s w e r e p r e i n c uba t e d w i t h a nt i m ous e C D 16/ C D 32 m onoc l ona l a nt i body ( c l one 2. 4G 2 P ha r M i nge n) f or 10 m i n a t 4C t o bl oc k non s pe c i f i c bi ndi ng t o F c r e c e pt or s C e l l s w e r e t he n s pun dow n a t 5, 000g w a s he d 3 t i m e s w i t h f l ow buf f e r a nd t he n i nc uba t e d w i t h ha m s t e r a nt i m ous e C D 40 F I T C or i s ot ype c ont r ol a nt i body F I T C ( 1: 100 di l ut i on; P ha r M i nge n, L os A nge l e s C A ) i n f l ow buf f e r A f t e r 30 m i n i nc uba t i on a t r oom t e m pe r a t ur e c e l l s w e r e w a s he d t w i c e w i t h f l ow buf f e r r e s us pe nde d i n 2 50 L of f l ow bu f f e r a nd a na l yz e d by a F A C S c a n T M i ns t r um e nt ( B e c t on D i c ki ns on, F r a nkl i n L a ne s N J ) A m i ni m um of 1 0, 000 c e l l s w e r e a c c e pt e d f o r F A C S a na l ys i s C e l l s w e r e ga t e d ba s e d on m or pho l ogi c a l c ha r a c t e r i s t i c s s uc h t ha t a popt ot i c a nd ne c r ot i c c e l l s w e r e not a c c e pt e d f o r F A C S a na l ys i s us i ng C e l l Q ue s t T M s of t w a r e ( B e c kt on D i c ki ns on) P e r c e nt a ge s of pos i t i ve c e l l s ( C D 40 e xpr e s s i ng) w e r e c a l c ul a t e d a s f ol l ow s : f or e a c h t r e a t m e nt t he m e a n f l uo r e s c e nc e va l ue f o r t he i s ot ype m a t c he d c ont r ol a nt i body w a s s ubt r a c t e d f r om t he m e a n f l uo r e s c e nc e va l ue f or t he C D 40 s pe c i f i c a nt i body 2. 6. 3 C D 40 p r ot e i n d e t e c t i on b y w e s t e r n b l ot F or w e s t e r n i m m unob l ot t i ng a na l ys i s of m i c r ogl i a l C D 40 e xpr e s s i on, f ol l ow i ng pa r a l l e l t r e a t m e nt c e l l l ys a t e s w e r e pr e pa r e d i n a n i c e c ol d l ys i s buf f e r ( 20 m M T r i s pH 7. 5, 150 m M N a C l 1 m M E D T A 1 m M E G T A 1 % T r i t on X 100, 2. 5 m M s odi um pyr ophos pha t e 1 m M gl yc e r o l phos pha t e 1 m M N a 3V O 4, 1 g/ m l l e upe pt i n a nd 1 m M\n20 P M S F ) a nd p r ot e i n c onc e nt r a t i on w a s de t e r m i ne d by t he B i o R a d pr o t e i n a s s a y a s pr e vi ous l y de s c r i be d ( T a n 2000) A n a l i quot c or r e s pondi ng t o 50 g of t ot a l p r ot e i n o f e a c h s a m pl e w a s s e pa r a t e d by S D S P A G E a nd t r a ns f e r r e d e l e c t r ophor e t i c a l l y t o i m m unobl ot t i ng P V D F m e m br a ne s N ons pe c i f i c a nt i body bi ndi ng w a s bl oc ke d w i t h 5% nonf a t dr y m i l k f or 1 h a t r oo m t e m pe r a t u r e i n T r i s buf f e r e d s a l i ne ( 20 m M T r i s a nd 500 m M N a C l pH 7. 5) S ubs e que nt l y, t he s e m e m br a ne s w e r e f i r s t hyb r i di z e d w i t h t he r a bbi t a nt i C D 40 a nt i body ( 1: 1 500 di l ut i on; S t r e s s G e n, V i c t or i a C a na da ) f o r 2 h a nd t he n w a s he d 3 t i m e s i n T B S a nd i m m unobl ot t i ng w a s by a n a nt i r a bbi t H R P c onj uga t e d I gG s e c onda r y a nt i body a s a t r a c e r T he l um i nol r e a ge nt w a s us e d t o de ve l op t he bl ot s T o de m ons t r a t e e qua l l oa di ng t he s a m e m e m br a ne s w e r e t he n s t r i ppe d w i t h m e r c a pt oe t ha nol s t r i ppi ng s ol ut i on ( 62. 5 m M T r i s H C l pH 6 8, 2% S D S a nd 100 m M m e r c a pt oe t ha nol ) a nd f i na l l y r e pr obe d w i t h m ous e m onoc l ona l a nt i body t o a c t i n. 2. 7 I m m u n ob l ot an al ys i s of t h e JA K / S T A T 1 s i gn al i n g p a t h w ay M ur i ne pr i m a r y c ul t ur e m i c r ogl i a l c e l l s w e r e pl a t e d i n 6 w e l l t i s s ue c ul t ur e pl a t e s a t a de ns i t y of 5 x 10 5 c e l l s pe r w e l l a nd i nc uba t e d w i t h I F N ( 100 U / m L ) i n t he pr e s e nc e or a bs e nc e of a dos e r a nge of l ova s t a t i n ( 2 5, 5. 0 10 0 a nd 20. 0 M ) f or 30 m i n I m m e di a t e l y a f t e r c ul t ur i ng m i c r ogl i a l c e l l s w e r e w a s he d i n i c e c ol d P B S t h r e e t i m e s a nd l ys e d i n a n i c e c ol d l ys i s buf f e r A f t e r i nc uba t i on f or 30 m i n on i c e s a m pl e s w e r e c e nt r i f uge d a t hi gh s pe e d f or 15 m i n, a nd s upe r na t a nt s w e r e c ol l e c t e d. T ot a l p r ot e i n c ont e nt w a s e s t i m a t e d us i ng t he B i o R a d pr ot e i n a s s a y. F or phos phor y l a t i on of J A K 1 a nd J A K 2, m e m br a ne s w e r e f i r s t hybr i di z e d w i t h phos pho s pe c i f i c T yr 1022/ 1023 J A K 1 or T y r 1007/ 1008 J A K 2 a nt i body a nd t he n s t r i ppe d a nd f i na l l y a na l yz e d by t ot a l J A K 1 or\n21 J A K 2 a nt i body. F or S T A T 1 a nd M A P K p42/ 44 p hos phor yl a t i on, m e m br a ne s w he r e f i r s t hybr i di z e d w i t h a phos pho s pe c i f i c p44/ 42 M A P K a nt i body, s t r i p pe d w i t h m e r c a pt oe t ha nol s t r i ppi ng s ol ut i on a nd t he n r e pr o be d w i t h a n a nt i body t ha t r e c ogni z e s t ot a l p44/ 42 M A P K O t he r m e m br a ne s w i t h i de nt i c a l s a m pl e s w e r e pr obe d w i t h a phos pho S e r 727 S T A T 1 a nt i body a nd s t r i ppe d w i t h s t r i ppi ng s ol ut i on a nd t he n r e p r obe d w i t h a n a nt i body t ha t r e c ogni z e s t ot a l S T A T 1 A l t e r na t i ve l y, m e m b r a ne s w i t h i de nt i c a l s a m pl e s w e r e pr obe d e i t he r w i t h phos pho J A K or S T A T 1 or w i t h a n a nt i body t ha t r e c ogni z e s t ot a l J A K or S T A T 1 I m m unobl ot t i ng w a s pe r f or m e d w i t h a pr i m a r y a nt i body f ol l ow e d by a n a nt i r a bbi t H R P c onj uga t e d I gG s e c onda r y a nt i body a s a t r a c e r A f t e r w a s hi ng i n T B S t he m e m br a ne s w e r e i nc uba t e d i n t he l um i nol r e a ge nt a nd e xpos e d t o f i l m 2. 8 S t at i s t i c al a n al ys i s D a t a a r e pr e s e nt e d a s m e a n + / S D A l l s t a t i s t i c s w e r e a na l yz e d us i ng a one w a y m ul t i pl e r a nge a na l ys i s of va r i a nc e t e s t ( A N O V A ) f or m ul t i pl e c om pa r i s ons A va l ue of p < 0. 05 w a s c ons i de r e d s i gni f i c a nt\n22 C H A P T E R T H R E E C D 45 I S O F O R M R B A S A M O L E C U L A R T A R G E T T O O P P O S E L P S I N D U C E D M I C R O G L I A L A C T I V A T I O N 3. 1 I n t r od u c t i on C D 45 i s a pr ot e i n t yr os i ne phos pha t a s e t ha t i s e xpr e s s e d on a l l nuc l e a t e d he m a t opoi e t i c c e l l s a nd pl a ys a n i m por t a nt r ol e i n r e gul a t i ng i m m une r e s pons e s B y de phos phor yl a t i ng S r c f a m i l y ki na s e s C D 45 pr om ot e s a nt i ge n s pe c i f i c B a nd T c e l l r e s pons e s ( P e nni nge r 2001; T hom a s 1999 ) C D 4 5 pl a ys a n a ddi t i ona l r ol e i n r e gul a t i ng s e l e c t i n e xpr e s s i on ( S t i be nz 1996; W r obl e w s ki 1 997) a nd i nt e g r i n f unc t i on ( R oa c h, 1997; S he noi 1999) R e c e nt l y, C D 45 ha s a l s o be e n s how n t o ne ga t i ve l y r e gul a t e c yt o ki ne r e c e pt or m e di a t e d s i gna l i ng vi a J A K f a m i l y ki na s e s t hus r e ve a l i ng a r ol e f or C D 45 i n m ul t i pl e he m a t opoi e t i c l i ne a ge s ( I r i e S a s a ki 2001) M ul t i pl e i s of or m s of C D 45 a r e ge ne r a t e d by a l t e r na t i ve s pl i c i ng of e xons 4, 5 a nd 6 ( a l s o know n a s A B a nd C ) t ha t e nc ode pa r t of t he e xt r a c e l l ul a r r e gi on o f t he m ol e c ul e H ow e ve r t he f unc t i ona l r ol e s of e a c h of t he C D 45 i s of o r m s on T c e l l s a s w e l l a s on ot he r nuc l e a t e d he m a t opoi e t i c c e l l s r e m a i ns unc l e a r ( B ot t om l y, 1989; D a vi e s 1999 ; T r ow br i dge 1994) S i m i l a r t o ot he r c e l l s of he m a t opoi e t i c l i ne a ge r e s t i ng m i c r ogl i a ha ve be e n s how n t o e xpr e s s C D 45 a t l ow t o m ode r a t e l e ve l s a nd C D 45 e xpr e s s i on m a r ke dl y i nc r e a s e s f ol l ow i ng t he i r a c t i va t i on ( C a r s on, 1998; S e dgw i c k, 1991) I n a ddi t i on, i t ha s be e n\n23 de m ons t r a t e d t ha t m i c r ogl i a l oc a t e d i n t he f r ont a l c or t e x a nd hi ppoc a m pus of nor m a l a gi ng i ndi vi dua l s e xpr e s s C D 45 a nd t hi s e xpr e s s i on l e ve l i s m a r ke dl y i nc r e a s e d i n A l z he i m e r s di s e a s e pa t i e nt s ( M a s l i a h, 1991) O ur pr e vi ous s t udi e s ha ve s how n t ha t c r os s l i nki ng C D 45 w i t h a n a goni s t i c a nt i C D 45 a nt i body m a r ke dl y i nhi bi t s C D 40L i nduc e d a c t i vi t y of t he S r c f a m i l y ki na s e s L c k a nd L yn. C o t r e a t m e nt of m i c r ogl i a w i t h C D 40L a nd a goni s t i c a nt i C D 45 a nt i body r e s ul t s i n s i gni f i c a nt i nhi bi t i on of m i c r ogl i a l T N F # pr oduc t i on vi a i nhi b i t i ng p44/ 42 M A P K a c t i vi t y, a dow ns t r e a m s i gna l i ng e ve nt r e s ul t i ng f r om S r c f a m i l y ki na s e a c t i va t i on ( T a n, 2 000) T he s e f i nd i ngs s ugge s t t ha t s t i m ul a t i on of t he C D 45 s i gna l i ng pa t hw a y m a y be di r e c t l y a ppl i c a bl e t o s uppr e s s i on of m i c r ogl i os i s a s s oc i a t e d w i t h ne ur ode ge ne r a t i ve di s or de r s I n or de r t o a ddr e s s w hi c h C D 45 i s of or m m a y m odul a t e m i c r ogl i a l a c t i va t i on f i r s t w e c ha r a c t e r i z e d t he C D 45 i s of or m pr o f i l e on r e s t i ng a nd a c t i va t e d m i c r ogl i a N e xt w e de t e r m i ne d i n v i t r o t he f unc t i on a l i t y of a goni s t i s of or m s pe c i f i c a nt i bodi e s t o m i t i ga t e L P S i nduc e d m i c r ogl i a l a c t i va t i on. F i na l l y, w e a na l yz e d t he t he r a pe ut i c po t e nt i a l of a C D 45R B s pe c i f i c a nt i body i n a n i n v i v o m ode l o f m i c r ogl i os i s P r i m a r y m ur i ne m i c r ogl i a l c e l l s w e r e obt a i ne d a n d c ul t ur e d a s de s c r i be d i n our pr e vi ous s t udi e s ( T a n, 1999) M i c r ogl i a l C D 45 i s o f or m e xpr e s s i on w a s a s s e s s e d by f l uor e s c e nc e a c t i va t e d c e l l s or t i ng ( F A C S ) a na l ys i s B r i e f l y c ul t u r e d m i c r ogl i a l c e l l s w e r e pl a t e d i n s i x w e l l t i s s ue c ul t ur e pl a t e s ( N unc l on T M ) a t 2 x 10 5 c e l l s / w e l l a nd i nc uba t e d i n t he p r e s e nc e or a bs e nc e of L P S T w e nt y f our hour s a f t e r i nc uba t i on, m i c r ogl i a l c e l l s ( a ppr ox i m a t e l y 1 x 10 6 c e l l s ) w e r e r e s us pe nde d i n 250 l o f i c e c ol d P B S f or F A C S a na l ys i s us i ng C D 45 i s of or m s pe c i f i c m onoc l ona l a nt i bodi e s ( pur i f i e d r a t a nt i m ous e pa n C D 45, pur i f i e d r a t a nt i m ous e C D 45R B a nd pur i f i e d I gG 2b c ont r o l\n24 a nt i bodi e s F I T C c onj uga t e d r a t a nt i m ous e pa n C D 45, F I T C c onj uga t e d r a t a nt i m ous e C D 45R A F I T C c onj uga t e d r a t a nt i m ous e C D 45R B F I T C c onj uga t e d r a t a nt i m ous e C D 45R C a nd F I T C c onj uga t e d r a t I gG 2b c ont r ol a nt i bodi e s ) pur c ha s e d f r om P ha r M i nge n ( S a n D i e go C A ) A m i ni m um of 10, 000 c e l l s w e r e a c c e pt e d f or F A C S a na l ys i s be i ng ga t e d ba s e d on m or phol og i c a l c ha r a c t e r i s t i c s s uc h t ha t a popt ot i c a nd ne c r ot i c c e l l s w e r e not a c c e pt e d. P e r c e nt a ge s of po s i t i ve ( C D 45 e xpr e s s i ng) c e l l s w e r e c a l c ul a t e d by s ubs t r a c t i ng t he m e a n f l uo r e s c e nc e va l ue f or t he i s ot ype m a t c he d c ont r ol a nt i body w a s s ubt r a c t e d f r om t he m e a n f l uor e s c e nc e va l ue f or t he C D 45 i s of or m s pe c i f i c a nt i body f or e a c h t r e a t m e nt I t s houl d be not e d t ha t be c a us e m i c r ogl i a c a n e xp r e s s m or e t ha n one C D 45 i s of or m a t onc e t hi s m e t hod of F A C S a na l ys i s onl y pe r m i t s a n a ppr oxi m a t e c om pa r i s on of C D 45 i s of or m e xpr e s s i on l e ve l s i n t e r m s of f l uor e s c e nc e br i ght ne s s a nd doe s not a l l ow f or s t r i c t qua nt i t a t i v e c om pa r i s on ( i e e a c h o f t he C D 45 i s of or m s w i l l not ne c e s s a r i l y s um t o t ha t of t he pa n C D 45) 3. 2 R e s u l t s 3. 2. 1 C D 45R B i s t h e m os t p r e val e n t C D 45 i s of o r m e xp r e s s e d b y e i t h e r r e s t i n g or ac t i vat e d m i c r ogl i a T o e xa m i ne f unc t i ona l i t y of C D 45 i s of or m s w e f i r s t e va l ua t e d w he t he r c r os s l i nki ng w i t h C D 45 i s of or m s pe c i f i c a nt i bodi e s ( a nt i C D 45R A a nt i C D 45R B or a nt i C D 45R C ) c oul d r e s ul t i n a n a goni s t i c ( i e s t i m ul a t or y) e f f e c t A s s t i m ul a t i on of C D 45 pr om ot e s t he r e c e pt or s t yr os i ne phos pha t a s e a c t i vi t y, w e m e a s ur e d f r e e i no r ga ni c i nt r a c e l l ul a r phos pha t e a s a n a c t i va t i on r e a dout a s pr e vi ous l y de s c r i be d ( T a n 2000) B r i e f l y, w e ut i l i z e d va r i ous C D 45 i s of or m s pe c i f i c a nt i bodi e s t o i m m unop r e c i pi t a t e t he i r\n25 r e s pe c t i ve t a r ge t s f r om m i c r ogl i a l c e l l l ys a t e s A f t e r a ddi t i on o f a phos pho s r c pe pt i de w e t he n m e a s ur e d t he a m ount of f r e e i no r ga ni c ph os pha t e r e l e a s e d by t he B i om ol G r e e n R e a ge nt A l l t he a nt i bodi e s t e s t e d w e r e e f f e c t i ve i n s t i m ul a t i ng t y r os i ne phos pha t a s e a c t i vi t y. T he pa n C D 45 a nt i body ha d a P T P a c t i vi t y ( nm ol / m i n) / ( O D / m m 2 ) of 0. 088 c om pa r e d t o 0 021, 0. 031 a nd 0 017 f or a nt i C D 4 5R A R B a nd R C r e s pe c t i ve l y, w he r e a s P T P a c t i vi t y w a s unde t e c t a bl e i n t he I gG i s ot ype a nt i body c ont r ol gr oup 3. 3. 2 L i gat i on of m i c r ogl i al C D 45R B an t agon i z e s L P S i n d u c e d m i c r ogl i al ac t i vat i on i n v i t r o H a vi ng e s t a bl i s he d t he a goni s t a c t i vi t y of t he va r i o us C D 45 a nt i bodi e s w e pr e t r e a t e d m i c r ogl i a w i t h e a c h of t he C D 45 a nt i bodi e s t he n c ha l l e nge d t he m w i t h L P S a nd e xa m i ne d T N F # pr oduc t i on a s a m a r ke r of t he i r a c t i va t i on. B r i e f l y, m i c r ogl i a l c e l l s w e r e pl a t e d i n 24 w e l l t i s s ue c ul t ur e pl a t e s ( C os t a r C a m br i dge M A ) a t 5 x 10 4 c e l l s pe r w e l l a nd s t i m ul a t e d f o r 18 h w i t h L P S ( 10 ng/ m l ; S i gm a ) i n t he pr e s e nc e or a bs e nc e of 2 h pr e t r e a t m e nt w i t h i s of o r m s pe c i f i c C D 45 a nt i bo di e s ( e a c h a t a 1: 200 di l ut i on) C e l l f r e e s upe r na t a nt s w e r e c ol l e c t e d a nd a s s a ye d f or T N F # us i ng a n E L I S A ki t ( B i os our c e I nt e r na t i ona l ) i n s t r i c t a c c or da nc e w i t h t he m a nuf a c t ur e r s i ns t r uc t i ons T o t a l p r o t e i n c ont e n t of t he c e l l l y s a t e w a s e s t i m a t e d us i n g t he B i o R a d pr ot e i n a s s a y ki t ( B i o R a d, H e r c ul e s C A ) T hus T N F # l e ve l s w e r e nor m a l i z e d t o t ot a l c e l l ul a r p r ot e i n a nd r e por t e d a s s uc h. 3. 3. 3 L i gat i on of m i c r ogl i al C D 45R B an t agon i z e s L P S i n d u c e d m i c r ogl i al ac t i vat i on i n v i v o\n26 W e f ur t he r w i s he d t o e va l ua t e t he pos s i bi l i t y t ha t C D 45R B m i ght be a ne ga t i ve r e gul a t or of m i c r ogl i a l a c t i va t i on i n v i v o by a dm i ni s t e r i ng a C D 45R B i s of or m s pe c i f i c a goni s t i c a nt i body t o m i c e c ha l l e nge d w i t h L P S A s w e r e qui r e d a n i ndus t r i a l qua nt i t y of a nt i body f or t he s e i n v i v o e xpe r i m e nt s w e f i r s t obt a i ne d a hybr i dom a c e l l l i ne ( A m e r i c a n T ype C ul t ur e C ol l e c t i on, M a na s s a s V A ) a nd s ubs e que nt l y c ul t ur e d t he s e c e l l s a nd pur i f i e d a nt i C D 45R B a nt i body ( c l one M G 23G 2) a s pr e vi ous l y de s c r i be d ( B a s a donna 1998; B i r ke l a nd, 1988) N i ne t e e n B A L B / C m i c e w e r e pur c ha s e d f r om J a c ks on L a bor a t or i e s ( B a r H a r bor M E ) F ol l ow i n g a t w o w e e k a c c l i m a t i on m i c e w e r e di vi de d i nt o t he f ol l ow i ng gr oups : g r oup 1 ( n = 5) r e c e i ve d 100 g o f L P S [ i n 100 L o f phos pha t e buf f e r e d s a l i ne ( P B S ) ] v i a a n i nt r a pe r i t o ne a l ( i p ) i nj e c t i on r ou t e ; gr oup 2 ( n= 4) r e c e i ve d a s e t of i p. pr e i nj e c t i ons w i t h M G 23G 2 ( 30 g pe r m ous e i n 100 L of vol um e i nj e c t e d) onc e a da y f o r t h r e e da ys be f or e t he L P S c ha l l e nge de s c r i be d a bove ; gr oup 3 ( n= 7) r e c e i ve d P B S ve hi c l e a l one ( 100 L ) ; l a s t l y, gr oup 4 ( n= 3 ) r e c e i ve d onl y t he s e t of p r e i nj e c t i ons w i t h M G 23G 2. A l l of t he m i c e w e r e s a c r i f i c e d 18 hou r s a f t e r t he l a s t i nj e c t i on by t r a ns c a r di a l pe r f us i on w i t h P B S u nde r i s of l uor a ne a ne s t he s i a I m m unohi s t oc he m i c a l de t e c t i on of C D 45 i n t he br a i n i s w i de l y ut i l i z e d a s a m i c r ogl i a l s pe c i f i c m a r ke r H ow e ve r de t a i l e d e xa m i na t i on of w hi c h C D 45 i s of or m s a r e e xpr e s s e d by m i c r ogl i a t he l e ve l s a t w hi c h t he y a r e e xpr e s s e d, a nd t he i r f unc t i on( s ) i s l a c ki ng. U t i l i z i ng F A C S a na l ys i s w e de m ons t r a t e t ha t t he C D 45R B i s of or m c ons t i t ut e s a ppr oxi m a t e l y 80% of t ot a l C D 45 e xpr e s s i on l e ve l on r e s t i ng m i c r ogl i a ( F i gur e 1) T o c ha r a c t e r i z e t he C D 45 i s of or m e xpr e s s i on pr of i l e i n a c t i va t e d m i c r ogl i a w e t he n e m pl o ye d t he pot e nt m i c r ogl i a l a c t i va t o r L P S M i c r ogl i a l c e l l s w e r e s t i m ul a t e d w i t h\n28 L P S ( 10 ng/ m l ) f or 18 hour s a nd F A C S a na l ys i s de m ons t r a t e s t ha t t he C D 45 i s of or m pr of i l e i s s i m i l a r t o t he r e s t i ng s t a t e ( w i t h C D 45 i s of or m R B c ons t i t ut i ng a ppr oxi m a t e l y 80% of t he t ot a l i s of or m s e xpr e s s e d) a l t hough t ot a l C D 45 e xpr e s s i on i nc r e a s e d f ol l ow i ng L P S t r e a t m e nt ( da t a not s how n) T o c ha r a c t e r i z e t he f unc t i on o f m i c r ogl i a l C D 45 i s of or m s i n t he c ont e xt o f a c t i va t i on, w e c ha l l e nge d m i c r og l i a w i t h a n L P S s t i m ul us i n t he p r e s e nc e of e a c h C D 45 i s of or m s pe c i f i c a goni s t a nt i body. E f f e c t s on m i c r ogl i a l T N F # pr oduc t i on a r e r e por t e d but I L 1 w a s a l s o m e a s ur e d ( da t a not s how n) a nd f ound t o r e pl i c a t e t he t r e nds s how n f or T N F # r e l e a s e A s e xpe c t e d, t h e pa n C D 45 i s of or m a nt i body ( F i gur e 2 c ol um n 3) m os t pot e nt l y s uppr e s s e d L P S i nduc e d T N F # pr o duc t i on ( 80% i nhi bi t i on ) H ow e ve r of t he t hr e e C D 45 i s of or m s pe c i f i c a nt i bodi e s us e d, o nl y a nt i C D 45R B w a s a bl e t o dr a m a t i c a l l y a nt a goni z e L P S i nduc e d m i c r o gl i a l a c t i va t i on, a s e vi de nc e d by a 70 75% r e duc t i on of T N F # ( F i gur e 2) T he s e da t a s ugge s t t ha t t a r ge t i ng C D 45R B on m i c r ogl i a m a y of f e r a n e f f e c t i ve a nd m or e s pe c i f i c a ppr oa c h t o s uppr e s s i ng a c t i va t i on ve r s us t ot a l C D 45. T o e xa m i ne t hi s hypot he s i s i n v i v o, w e e va l ua t e d w he t he r m i c e pr o phyl a c t i c a l l y t r e a t e d w i t h t he C D 45R B i s of or m s pe c i f i c a nt i body M G 23G 2, w oul d be ne f i t i n t e r m s of m i t i ga t i ng t he m i c r ogl i os i s nor m a l l y a s s oc i a t e d w i t h i p. L P S c ha l l e nge D a t a s how t ha t br a i n hom oge na t e s f r om L P S i n j e c t e d m i c e t ha t r e c e i ve d pr e t r e a t m e nt w i t h M G 23G 2 e xpr e s s s i gni f i c a nt l y l e s s T N F # c om pa r e d t o m i c e t ha t di d not r e c e i ve t he pr e t r e a t m e nt ( F i gu r e 3)\n31 3. 4 D i s c u s s i on C hr oni c m i c r ogl i a l a c t i va t i on ha s be e n i m pl i c a t e d a s a pa t hoge ni c f a c t or i n a va r i e t y of ne ur ode ge ne r a t i ve di s e a s e s s uc h a s m ul t i pl e s c l e r os i s A I D S de m e nt i a a nd A l z he i m e r s di s e a s e r a i s i ng t he pos s i bi l i t y t ha t t he r a pe ut i c s t r a t e gi e s a i m e d a t oppos i ng m i c r ogl i a l a c t i va t i on m a y be be ne f i c i a l i n t r e a t i ng s uc h di s e a s e s H ow e ve r c u r r e nt a t t e m pt s a t r e duc i ng ne ur oi n f l a m m a t i on m e di a t e d vi a c hr oni c m i c r og l i a l a c t i va t i on ha ve onl y be e n pa r t i a l l y e f f i c a c i ous ( R i c h, 1995) pos s i bl y be c a us e of t he f a c t t ha t s uc h s t r a t e gi e s a r e be t t e r c ha r a c t e r i z e d a s ge ne r a l i nhi bi t or s of i n f l a m m a t i on t ha n t he y a r e s pe c i f i c i nhi bi t or s of m i c r ogl i a l a s s oc i a t e d i nf l a m m a t i on. I n t hi s s t udy, w e f oc us e d on c ha r a c t e r i z i ng va r i ous i s of or m s o f a m i c r ogl i a s ur f a c e r e c e pt or t a r ge t C D 45, w hi c h w e ha ve pr e vi ous l y s how n i s a bl e t o a nt a goni z e m i c r o gl i a a c t i va t i on ( T a n, 2000a b) C D 45 i s of or m s ha ve be e n de s c r i be d on ot he r c e l l l i ne a ge s but t hi s i s t he f i r s t t i m e t o our know l e dge t ha t t he y ha ve be e n c ha r a c t e r i z e d on m i c r ogl i a c e l l s W e r e por t he r e i n t ha t C D 45R B i s t he pr i nc i pl e i s of or m of C D 45 on m u r i ne m i c r ogl i a e i t he r w he n r e s t i ng or f ol l ow i ng L P S a c t i va t i on T hi s l a t t e r obs e r va t i on i s i nt e r e s t i ng, a s i t s ugge s t s t ha t unl i ke T c e l l s C D 45 i s of o r m s w i t c hi ng doe s not oc c ur f o l l ow i ng m i c r ogl i a l a c t i va t i on. W e f ur t he r de m ons t r a t e t ha t a c t i va t i on of t he C D 45R B i s of or m w i t h a n i s of or m s pe c i f i c a nt i body i s a bl e t o pot e nt l y oppos e L P S i nduc e d m i c r ogl i a l a c t i va t i on i n v i t r o F i na l l y w e s how i n v i v o t ha t a n a goni s t i c a nt i C D 45R B a nt i body i s a bl e t o a t t e nua t e C N S i nf l a m m a t i on i nduc e d by i p. i nj e c t i on of L P S S p e c i f i c a l l y, w e f i nd t ha t a pr ophy l a c t i c t r e a t m e nt r e gi m e n w i t h a nt i C D 45R B a nt i body i s a bl e t o m i t i ga t e L P S i nduc e d T N F # pr ot e i n l e ve l s i n t he br a i n A s m i c r ogl i a a r e t he on l y C N S c e l l popul a t i on t ha t e xp r e s s e s C D 45, i t i s r e a s ona bl e t o a s s um e t ha t t hi s a nt i body i s e xe r t i ng i t s e f f e c t on m i c r ogl i a\n32 A nt i bodi e s a ga i ns t di f f e r e nt C D 45 i s of or m s or di f f e r e nt e pi t ope s of t he s a m e i s of or m c a n e l i c i t di f f e r e nt bi ol ogi c a l e f f e c t s a nd onl y s om e ha ve be e n s how n t o pr ol on g a l l ogr a f t s ur vi va l ( F a br e 1977; G a o, 1999; G ol dbe r g 1995; L a z a r ovi t s 1996; R ot hs t e i n, 2001) T he a nt i C D 45R B a nt i body w e e m pl oye d ( M G 23G 2) ha s be e n s how n t o p r om ot e t he e s t a bl i s hm e nt of a l l ogr a f t s a nd xe nog r a f t s ( B a s a donna 1998; L a z a r ovi t s 1996 ) T hi s r e s ul t i s c ons i s t e nt w i t h t he i de a t ha t M G 23G 2 opp os e s i m m une c e l l a c t i va t i on not on l y i n t he C N S a s w e r e por t but a l s o i n t he pe r i phe r y T a ke n t oge t he r t he s e da t a s how t ha t s t i m ul a t i on of t he C D 45 pa t hw a y, s pe c i f i c a l l y t he C D 45R B i s of or m ne ga t i ve l y c ont r ol s m i c r ogl i a l a c t i va t i on i nduc e d by pr oi nf l a m m a t or y s t i m ul a t i on. A s ugge s t i on t ha t a r i s e s f r om t he s e r e s ul t s i s t ha t pha r m a c ot he r a pe ut i c s t ha t s pe c i f i c a l l y s t i m ul a t e C D 45R B m a y be be ne f i c i a l f o r m i t i ga t i ng m i c r ogl i os i s a s s oc i a t e d w i t h ne ur ode ge ne r a t i ve di s e a s e s\n33 C H A P T E R F O U R L O V A S T A T I N M O D U L A T I O N O F M I C R O G L I A L A C T I V A T I O N V I A S U P P R E S S I O N O F C D 40 A N D I T S S I G N A L I N G P A T H WA Y 4. 1 I n t r od u c t i on S t a t i ns a r e e f f e c t i ve l i pi d l ow e r i ng a ge nt s t ha t i nh i bi t t he r a t e l i m i t i ng e nz ym e of c hol e s t e r ol bi os ynt he s i s 3 hydr oxy 3 m e t hyl gl ut a r yl c oe nz ym e A r e duc t a s e ( H M G C oA ) I nt e r e s t i ngl y s t a t i ns ha ve s how n c l i ni c a l e f f i c a c y not onl y i n t he t r e a t m e nt o f a r t e r i os c l e r os i s a nd hype r c hol e s t e r ol e m i a ( S he phe r d, 1995; K ui ve nhove n, 1998; G a r l i c hs 2001 a, b ; A r onow 2003 ; S e m b, 2003) bu t a l s o i n t he t r e a t m e nt o f ne ur ode ge ne r a t i ve di s e a s e s l i ke A l z he i m e r s D i s e a s e ( A D ) ( W ol oz i n 2000; S i m ons 2002 ; K i r s c h 2003; Z a m r i ni 2004) H ow e ve r a num be r o f s t udi e s ha ve s how n t ha t t he e f f i c a c y of c e r t a i n s t a t i ns a ppe a r t o be di s s oc i a t e d f r om e f f e c t s on H M G C oA i nhi bi t i on s ugge s t i ng t ha t s t a t i ns di s pl a y p l e i ot r oph i c e f f e c t s out s i de of t he i r l i pi d l ow e r i ng pr ope r t i e s ( L e f e r 2001; M a r z 2003 ) I n f a c t r e por t s o f t he e f f e c t s of s t a t i ns on va s c ul a r de r i ve d c e l l s s ugge s t t ha t t he y s uppr e s s C D 40 e xpr e s s i on a nd s ubs e que nt i nf l a m m a t or y p r oc e s s e s ( S c honbe c k, 2 002; W a gn e r 2002; M ul ha upt 2003 ) C D 40 i s a 50 kD a t ype I phos phopr ot e i n t ha t i s a m e m be r of t he T N F r e c e pt or s upe r f a m i l y. C D 40 i s e xpr e s s e d by a w i de va r i e t y of c e l l s ( S c hnbe c k, 2001) T he l i ga nd f or C D 40 ( C D 154) i s e xpr e s s e d m a i nl y on a c t i va t e d C D 4+ T c e l l s a l t hough ot he r c e l l s s uc h a s va s c ul a r e ndot he l i a l c e l l s s m oot h m u s c l e c e l l s m a c r opha ge s a nd a s t r oc yt e s\n34 ha ve t he c a pa c i t y t o e xpr e s s C D 154. U pon l i ga t i o n of C D 40, num e r ous c e l l t ype de pe nde nt s i gna l i ng pa t hw a ys a r e a c t i va t e d, l e a di n g t o c ha nge s i n ge ne e xp r e s s i on a nd f unc t i on. T he s e c ha nge s i nc l ude N F kB m i t oge n a c t i va t e d pr ot e i n ( M A P ) ki na s e s T N F R a s s oc i a t e d f a c t or pr ot e i ns P I 3K a nd t he J A K / S T A T pa t hw a y ( va n K oot e n 2000 a, b ) L i ga t i on of C D 40 on m i c r ogl i a l c e l l s l e a ds t o t he pr oduc t i on o f T N F # a nd ot he r uni de nt i f i e d ne ur ot oxi ns ( A l oi s i 1999; F i s c h e r 2001; D A ve r s a 2002 ) T hus s i gna l i ng t hr ough C D 40 on m i c r ogl i a l c e l l s i nduc e s a num be r of s ol ubl e m e di a t or s t ha t ha ve i m por t a nt f unc t i ona l r o l e s i n t he c e nt r a l ne r v ous s ys t e m ( C N S ) I n t he no r m a l b r a i n, m i c r ogl i a d i s pl a y a qui e s c e nt phe not ype i nc l udi ng l ow C D 40 e xpr e s s i on ( T a n, 1999 a ) H ow e ve r upon i ns ul t t o t he br a i n, m i c r ogl i a be c om e hi ghl y a c t i va t e d, a l t e r i ng t he i r pha goc yt i c a nd a nt i ge n p r e s e nt a t i on f unc t i ons ( T ow ns e nd, s ubm i t t e d 2004) a s w e l l a s t he pr oduc t i on of c yt oki ne s ( A l oi s i 2001) M ount i ng e vi de nc e i m pl i c a t e s m i c r ogl i a l C D 40 a s c ont r i but i ng t o t he i ni t i a t i on a nd/ or p r ogr e s s i on of s e ve r a l ne ur ode ge ne r a t i ve di s e a s e s i nc l udi ng: H I V 1 a s s oc i a t e d de m e nt i a ( D 'A ve r s a 2002) m u l t i pl e s c l e r os i s ( M S ) ( G r e w a l 1996; L a m a n, 1998a ; B e c he r 2001; N a t h 2004 ) a nd A D ( T ogo 2000 ; O 'K e e f e 2002; C a l i nga s a n, 2002) I n f a c t bl oc ki ng C D 40 C D 154 i nt e r a c t i ons by a ne ut r a l i z i ng a nt i body s t r a t e gy pr e ve nt s m ur i ne e xpe r i m e nt a l a ut oi m m une e nc e pha l om ye l i t i s ( E A E ) d i s e a s e a c t i vi t y ( G e r r i t s e 1996; L a m a n, 1998 b ; B oon, 2001; H ow a r d 2003 ) a s w e l l a s A D l i ke pa t hol ogy i n m ous e m ode l s o f t he di s e a s e ( T a n, 2002) G i ve n t he e pi de m i ol ogi c a l da t a s uppor t i ng t he us e of s t a t i ns i n A D a nd t he r e c e nt w or k s how i ng t ha t s t a t i ns oppos e C D 40 e xpr e s s i on i n va s c ul a r de r i ve d c e l l s w e now e xa m i ne w he t he r s t a t i ns ( s pe c i f i c a l l y l ova s t a t i n a l s o r e f e r r e d t o a s m e vi nol i n ) c oul d\n35 oppos e m i c r ogl i a l C D 40 e xpr e s s i on a nd i t s s i gna l i ng pa t hw a y f ol l ow i ng I F N c ha l l e nge F ur t he r m or e w e e xa m i ne w he t he r l ova s t a t i n i n f l u e nc e s m i c r ogl i a l pha goc yt i c f unc t i on a nd/ or pr o i nf l a m m a t or y c yt oki ne p r oduc t i on a f t e r C D 40 l i ga t i on. 4. 2 R e s u l t s 4. 2. 1 L ovas t at i n i n h i b i t s I F N an d A p e p t i d e i n d u c e d C D 40 e xp r e s s i on i n m i c r ogl i a A l t hough pr e vi ous s t udi e s ha ve s how n t ha t C D 40 e xpr e s s i on i s c ons t i t ut i ve l y e xpr e s s e d a t l ow l e ve l s i n m i c r ogl i a l c e l l s ; C D 40 e xpr e s s i on i s dr a m a t i c a l l y i nduc e d i n r e s pons e t o I F N or A pe pt i de c ha l l e nge ( N guye n, 1998; T a n, 1999 a, b ) I n o r de r t o e xa m i ne l ova s t a t i n r e gul a t i on of C D 40 e xpr e s s i on i n m i c r ogl i a l c e l l s c ul t ur e d m ous e m i c r ogl i a l c e l l s w e r e t r e a t e d w i t h I F N ( 100 U / m L ) f or 3 h a nd m R N A s f or C D 40 w e r e a na l yz e d by s e m i qua nt i t a t i ve R T P C R C ons i s t e nt w i t h ou r pr e vi ous da t a a nd t he da t a of ot he r s I F N c ha l l e ng e p r oduc e d a r obus t i nc r e a s e i n C D 40 m R N A e xpr e s s i on ( F i gur e 4) L ova s t a t i n t r e a t m e nt ( 2 5 t o 20 M ) di d not a f f e c t c ons t i t ut i ve C D 40 e xpr e s s i on ( F i gur e 4 l a ne 5 f r om l e f t ) but t he 10 M dos e m a r ke dl y i nhi bi t e d I F N i nduc e d C D 40 e xpr e s s i on ( F i gur e 4 l a ne 10 c om pa r e d t o l a ne 7 f r om l e f t ) I nt e r e s t i ngl y unde r t he s a m e t r e a t m e nt c ondi t i ons I F N i nduc e d C D 40 m R N A e xpr e s s i on w a s not a f f e c t e d by e i t he r t he M A P K i nhi bi t or s ( P D 98059 o r S B 203580) f o r p42/ 44 M A P K or p38 M A P K r e s pe c t i ve l y ( da t a not s how n) T o e xa m i ne w he t he r l ova s t a t i n s i nhi bi t i on of C D 40 m R N A l e d t o a s i m i l a r r e duc t i on i n p r ot e i n e xpr e s s i on, w e s t e r n i m m unobl ot a nd F A C S a na l ys e s w e r e pe r f or m e d on p r i m a r y m ous e m i c r o gl i a l c e l l s a nd t he N 9 m i c r ogl i a l c e l l s l i ne ( da t a not s how n) c ul t ur e d i n t he pr e s e nc e of I F N ( 100 U / m L ) f or 12 h I F N\n37 pr oduc e d a s ubs t a nt i a l i nc r e a s e i n C D 40 e xpr e s s i on a s m e a s ur e d by w e s t e r n i m m unobl ot t i ng ( F i gur e 5 a ) a nd qua nt i t a t i ve c or r e c t e d m e a n f l uor e s c e nc e uni t s ( F i gu r e 5 b ) I n bot h c a s e s l ova s t a t i n c o t r e a t m e nt m i t i ga t e d t he i nduc i bl e i nc r e a s e i n C D 40 pr ot e i n e xpr e s s i on i n p r i m a r y c ul t u r e d m i c r ogl i a l c e l l s ( F i gur e 5 a b ) T a ke n t oge t he r t he s e f i ndi ngs de m ons t r a t e t ha t l ova s t a t i n oppos e s I F N i nduc e d C D 40 e xpr e s s i on on pr i m a r y c ul t u r e d m i c r ogl i a l c e l l s 4. 2. 2 L ovas t at i n i n h i b i t s JA K / S T A T s i gn al i n g p at h w ay i n d u c e d b y I F N i n m i c r ogl i a A s pr e vi ous l y m e nt i one d, I F N i s a pot e nt i nduc e r of m i c r ogl i a l C D 40 e xpr e s s i on a nd t he s i gna l t r a ns duc t i on pa t hw a y i nv ol ve d i n t hi s i nduc t i on ha s be e n de s c r i be d by m a ny t o i nvol ve e l e m e nt s of t he J A K / S T A T s i gna l i ng pa t hw a y. I nt e r e s t i ngl y m a ny of t he f a c t or s ( c yt oki ne s ne ur ot r ophi ns ne ur ope pt i de s ) t ha t i nhi bi t I F N i nduc e d m i c r og l i a l C D 40 e xpr e s s i on do s o b y m odi f i c a t i on of t he J A K / S T A T pa t hw a y N guye n, 2000 a, b ; W e i 1999; D e l ga do 2 003; K i m 2002) T he r e f o r e w e e xa m i ne t he e f f e c t s of l ova s t a t i n on t he J A K / S T A T s i gna l i ng pa t hw a y i n c ul t ur e m ous e m i c r ogl i a l c e l l s M ous e m i c r ogl i a l c e l l s w e r e t r e a t e d w i t h I F N f or 30 m i n i n t he pr e s e nc e or a bs e nc e of a dos e r a nge o f l ova s t a t i n ( 0 20 M ) W e s t e r n i m m unobl ot t i ng a na l ys i s r e ve a l e d t ha t l ova s t a t i n m a r ke dl y m i t i ga t e d J A K 1 T yr 1022/ 1023 a nd J A K 2 T yr 1007/ 1008 phos phor yl a t i on a t 10 M dos e ( F i g ur e 6 a pa ne l 1 t o 4) H ow e ve r t he phos phor yl a t i on of J A K 1 a nd J A K 2 w a s onl y s l i gh t l y i nhi bi t e d by l ova s t a t i n a t 5 M F ur t he r m or e i t i s w e l l know n t ha t du r i ng I F N i nt e r a c t i on w i t h i t s he t e r odi m e r t ype I I c yt oki ne r e c e pt or t he J A K s a r e di r e c t l y a c t i va t e d l e a di ng t o S T A T 1 phos phor yl a t i on\n40 ( N guye n, 2000 a b ; D e l ga do, 2003) A c c or di ngl y w e e xa m i ne d t he e f f e c t s of l ova s t a t i n on S T A T 1 phos phor yl a t i on i n t he s a m e dos e r a ng e m e nt i one d a bove on pr i m a r y m i c r ogl i a l c e l l s t r e a t e d w i t h I F N f or 30 m i n. D a t a s how e d t ha t l ova s t a t i n s i gni f i c a nt l y i nhi bi t e d S e r 727 phos phor yl a t i on o f t he S T A T 1 pr ot e i n a t 10 M ( F i gu r e 7 pa ne l s 1 a nd 2) ; c ons i s t e nt w i t h t he e f f e c t s on J A K phos phor yl a t i on, 5 M of l ova s t a t i n w a s l e s s e f f e c t i ve T he M A P K pa t hw a y ha s be e n r e por t e d t o m e di a t e f ul l t r a ns c r i pt i on a c t i va t i on of S T A T 1 by t he phos phor yl a t i on o f S e r 727 ( G oh 1999; D a l pke 20 03 ) W e t he r e f or e i nve s t i ga t e d w he t he r p42/ 44 M A P K phos phor yl a t i on s t a t us r e f l e c t e d t ha t o f S T A T 1. D a t a r e ve a l e d t ha t t he r e w a s no c ha nge i n p42/ 44 M A P K phos phor yl a t i on s t a t us i n m i c r ogl i a l c e l l s t r e a t e d w i t h I F N f or 30 m i n i n t h e a bs e nc e or pr e s e nc e of l ova s t a t i n ( F i gur e 7 pa ne l 3 a nd 4 ) T a ke n t oge t he r t he s e obs e r va t i ons s ugge s t t ha t l ova s t a t i n oppos e s I F N i nduc t i on of t he J A K / S T A T pa t hw a y i n m i c r ogl i a l c e l l s w i t hout a f f e c t i ng p42/ 44 M A P K phos phor yl a t i on. 4. 2. 3 L ovas t at i n i n h i b i t s f u n c t i on al C D 4 0 s i gn al i n g i n m i c r ogl i a T o e xa m i ne f unc t i ona l c ons e que nc e s of l ova s t a t i n t r e a t m e nt on C D 40 e xpr e s s i on, w e s t i m ul a t e d m ous e pr i m a r y m i c r ogl i a l c e l l s w i t h e i t he r I F N / a goni s t a nt i C D 40 a nt i body or A 1 $ 4 2 / a goni s t a nt i C D 40 a nt i body i n t he pr e s e nc e or a bs e nc e of l ova s t a t i n f or 12 h. S upe r na t a nt s f r om e a c h t r e a t m e nt c ondi t i on w e r e e xa m i ne d by E L I S A f or a pa ne l of pr o i nf l a m m a t o r y m ol e c ul e s t ha t w e ha ve pr e vi ous l y de s c r i be d a s be i ng i nduc e d by m i c r ogl i a l C D 40 l i ga t i on ( T a n 1999 a b, c ; 2000 ) A s w e e xpe c t e d, E L I S A m e a s ur e m e nt s r e ve a l e d t ha t e i t he r I F N / a goni s t a nt i C D 40 a nt i body o r A 1 $ 4 2 / a goni s t\n42 a nt i C D 40 a nt i body i nduc e d t he s e c r e t i on of t he pr o i nf l a m m a t or y m ol e c ul e s I L 6, I L 1 a nd T N F # a s i ndi c a t e d i n F i gur e s 8 10 H ow e ve r w he n s t i m ul a t e d i n t he pr e s e nc e of l ova s t a t i n, a l l of t he s e pr o i nf l a m m a t or y pr ot e i ns w e r e s i gni f i c a nt l y r e duc e d ( F i gur e s 8 10) T he c oni c a l m i c r ogl i a f unc t i on i n t he C N S i s t hought t o be pha goc yt os i s a nd gi ve n t ha t I F N a nd C D 40 s i gna l i ng a r e m a t u r a t i on a ge nt s t ha t oppos e t hi s pha goc yt i c f unc t i on ( A l oi s i 2000; M a gnus 2001; C ha n, 2001; R e 20 02; M ons one go, 2003 a b ) w e e xa m i ne d w he t he r l ova s t a t i n c o t r e a t m e nt c oul d r e s c ue m i c r ogl i a pha goc yt i c f unc t i on P r i m a r y m ous e m i c r ogl i a l c ul t ur e s w e r e e xpos e d t o 3 M o f A 1 $ 4 2 ( f or i m m unobl ot t i ng) or C y3 % A 1 4 2 ( f or pha goc yt o s i s a s s a y) i n t he pr e s e nc e or a bs e nc e of a goni s t a nt i C D 40 a nt i body or a goni s t a nt i C D 40 a nt i body/ l ova s t a t i n. A f t e r 3 h, t he a m ount o f pha goc yt os e d A pe pt i de w a s de t e r m i ne d by bot h qua l i t a t i ve i m m unof l uo r e s c e nc e s t udi e s ( F i gur e 11) a nd w i t h qua nt i t a t i ve i m m unob l ot t i ng e xpe r i m e nt s ( F i gur e 12 ) A s s how n i n F i gur e 11, a goni s t a nt i C D 40 a nt i body pr e t r e a t m e nt de c r e a s e d m i c r ogl i a pha goc yt i c f unc t i on c om pa r e d t o c ont r ol s ( pa ne l 1 ve r s us 3 or 4) w hi l e l ova s t a t i n t r e a t m e nt a l one di d not di f f e r f r om t ha t o f c ont r ol ( pa ne l 1 ve r s us 2) I nt e r e s t i ngl y, t he pr e s e nc e of l ova s t a t i n r e s c ue d m i c r ogl i a l pha goc yt os i s of A f ol l ow i ng a goni s t a nt i C D 40 a nt i body t r e a t m e nt ( F i gur e 11, pa ne l 4 ve r s us 3) I n a pa r a l l e l e xpe r i m e nt w e f ur t he r s how e d t ha t t he p r e s e nc e s of l ova s t a t i n r e s ul t e d i n a s i gni f i c a nt a t t e nua t i on o f i m pa i r e d m i c r ogl i a l pha goc yt os i s of A i nduc e d b y a goni s t a nt i C D 40 a nt i body a s e vi de nc e d by i nc r e a s e d ba nd de ns i t y r a t i o o f A t o a c t i n us i ng w e s t e r n i m m unob l ot t i ng ( F i gur e 12 )\n48 4. 3 D I S C U S S I O N T he pr e s e nt f i ndi ngs de m ons t r a t e t ha t s t a t i ns na m e l y l ova s t a t i n, r e duc e I F N i nduc e d upr e gul a t i on of C D 40 e xp r e s s i on i n m ous e m i c r ogl i a l c e l l s by i nt e r f e r i ng w i t h t he J A K / S T A T pa t hw a y G i ve n t ha t t hi s f i ndi ng i s c ons i s t e nt w i t h t he e f f e c t s of l ova s t a t i n i n I F N t r e a t e d va s c ul a r de r i ve d c e l l s ( W a gne r 2002; S c honbe c k, 2002; M ul ha upt 2003) t he s i gni f i c a nc e of l ova s t a t i n bl o c ki ng I F N i nduc e d m i c r ogl i a l C D 40 e xpr e s s i on m us t be c ons i de r e d i n t he c ont e xt of t h e f unc t i on o f m i c r og l i a l C D 40. A be r r a nt e xpr e s s i on of C D 40 by m a c r opha ge s / m i c r ogl i a i n c onj unc t i on w i t h t he r e l e a s e of T N F # i s di r e c t l y c or r e l a t e d w i t h p a t hoge ni c e ve nt s oc c ur r i ng i n t he C N S i n t he di s e a s e of m ul t i pl e s c l e r os i s ( W a gne r 2002; S c ho nbe c k, 2002; M ul ha upt 2003) I t i s a l s o c l e a r t ha t C D 40 e xpr e s s i on by m i c r ogl i a l c e l l s i s pi vot a l f or di s e a s e i ni t i a t i on a nd pr ogr e s s i on i n t he E A E m ous e m ode l of M S ( B e c he r 2001 ) I n A D a c t i va t e d m i c r ogl i a a r e c ons i de r e d t o be a m a j or c ont r i but or t o t he l oc a l i nf l a m m a t or y r e s pons e s e vi de nc e d i n ne ur i t i c p l a que s F u r t he r m o r e t he C D 40 C D 40 L dya d i s po t e nt i a t e d a s s e e n by t he i nc r e a s e d C D 40 pos i t i ve m i c r ogl i a a s w e l l a s i nc r e a s e d C D 40L e xpr e s s i on on a s t r oc yt e s i n A D ( C a l i nga s a n, 2002 ) O ur pr e vi ous w or k ha s s how n a c or r e l a t i on be t w e e n i nc r e a s e d l e ve l s of A pe pt i de a nd e nha nc e d C D 40 e xpr e s s i on on m i c r ogl i a de r i ve d f r om t he T g2576 m ous e m ode l o f A D ( T a n e t a l 1 999 a ) W e ha ve a l s o s how n t ha t A c ha l l e nge i nduc e s m i c r ogl i a l C D 40 e xpr e s s i on, t he l i ga t i on o f w hi c h r e s ul t e d i n t he pr oduc t i on of m i c r ogl i a l de r i ve d ne u r ot oxi ns ( T a n, 1999 a ) I n v i v o va l i da t i on of a pa t hoge ni c r ol e f o r m i c r ogl i a l C D 40 w a s s how n b y t he be ne f i c i a l e f f e c t s of s t r a t e gi e s ( C D 40 ne ut r a l i z i ng a nt i bodi e s o r C D 40L ge ne t i c k noc kout s ) i nvol vi ng t he bl oc ka de of C D 40 s i gna l i ng i n A D m ous e m ode l s ( T a n, 2002)\n49 I n a ddi t i on t o t he e pi de m i ol ogi c a l s t udi e s s ugge s t i ng t ha t s t a t i n us e i s a s s oc i a t e d w i t h a l ow e r i nc i de nc e of A D a num be r o f r e por t s ha ve a l s o s how n t ha t l ova s t a t i n c a n r e duc e pl a s m a A l e ve l s i n A D pa t i e nt s ( F r i e dhof f 2001; B uxba um 2002 ) a nd t he i nc r e a s e d r e por t s of i t s H M G C oA i nde pe nde nt pr ope r t i e s O ur s t udy s ugge s t s t ha t one of l ova s t a t i n s m e c ha ni s m s of a c t i on m a y be t he a t t e nua t i on of pa t hoge ni c m i c r ogl i a l C D 40 e xpr e s s i on. T w o ot he r s t a t i ns a t or va s t a t i n a nd c e r i va s t a t i n, w e r e p r e vi ous l y s how n t o i nhi bi t C D 40 e xpr e s s i on i n m onoc yt e s a n d m a c r opha ge s a l t hough t he m e c ha ni s m of a c t i on w a s not de t e r m i ne d ( G a r l i c hs 2001; Y ous s e f 2002) A r e vi e w of t he m ol e c ul a r ba s i s of C D 40 e xp r e s s i on i n m a c r op ha ge s / m i c r ogl i a i l l um i na t e s t he c r i t i c a l r ol e of t he J A K / S T A T pa t hw a y ( B e nve ni s t e 2004) I n t hi s s t udy, w e s how t ha t l ova s t a t i n i nhi bi t s I F N i nduc e d m i c r ogl i a l C D 40 e xpr e s s i on by oppos i ng J A K / S T A T pa t hw a y a c t i va t i on. O ne pos s i bl e m e c ha ni s m of l o va s t a t i n s i nhi bi t i on of t he J A K / S T A T pa t hw a y i s pr ovi de d by a r e c e nt r e por t s how i ng t h a t i n m a c r opha ge s l ova s t a t i n t r e a t m e nt i nduc e d s uppr e s s or of c yt oki ne s i gna l i ng p r ot e i n ( S O C S ) w hi c h unc oupl e d I F N f r om S T A T a c t i va t i on ( H ua ng, 2003) M or e ove r c ons t i t ut i ve ove r e xpr e s s i on of t he S O C S 1 pr ot e i n i n t he m ur i ne m a c r opha ge c e l l l i ne R A W 264. 7, ha s be e n s how n t o i nhi bi t I F N but not L P S i nduc e d C D 40 e xpr e s s i on ( W e s e m a nn, 2002) T he S O C S pr o t e i ns a c t a s ne ga t i ve r e gul a t or s of t he J A K / S T A T pa t hw a y e i t h e r by bi ndi ng a nd i nhi b i t i ng J A K t yr os i ne ki na s e s or by i nhi bi t i ng bi ndi ng of S T A T f a c t or s t o t he c yt opl a s m i c dom a i ns of t he r e s pe c t i ve r e c e pt or s ( Y a s uk a w a 2000) I nt e r e s t i ngl y, i n t he s t udy by H ua ng, a 10 M dos e of l ova s t a t i n di d not i nduc e m a c r opha ge S O C S 3 pr ot e i n e xpr e s s i on unt i l 16 h a f t e r t r e a t m e nt I n our s t udy how e ve r l ova s t a t i n a t t he s a m e dos e i nhi bi t e d no t onl y J A K / S T A T phos phor yl a t i on but a l s o C D 40 m R N A w i t hi n 30 m i n a nd 3 h r e s pe c t i ve l y.\n50 T hi s s ugge s t s t ha t l ova s t a t i n s a c ut e e f f e c t s on I F N s i gna l i ng do not i nvol ve S O C S 3 W e c a nnot how e ve r r ul e out a r ol e f or S O C S 3 i n duc t i on by l ova s t a t i n i n a br oga t i on of I F N i nduc e d m i c r og l i a l pr o i nf l a m m a t o r y c yt oki ne pr oduc t i on o r pha goc yt i c f un c t i on be c a us e of t he l onge r t r e a t m e nt t i m e s e m pl oye d i n t hos e a s s a y s W e ha ve pr e vi ous l y s how n t ha t a nt a goni z i ng m i c r ogl i a l C D 40 e xpr e s s i on or C D 40 dow ns t r e a m s i gna l i ng pa t hw a ys c oul d a l s o bl oc k m i c r ogl i a l p r oduc t i on o f p r o i nf l a m m a t or y m e di a t o r s ( T a n, 20 00 ; 2002) I n t hi s s t udy, w e ha ve s how n t ha t l ova s t a t i n s i m i l a r l y i nhi bi t e d m i c r ogl i a l C D 40 l i ga t i on i nduc e d pr oi nf l a m m a t o r y c yt oki ne pr oduc t i on. T hi s f i ndi ng i s c ons i s t e nt w i t h ot he r r e por t s of l ova s t a t i n i nh i bi t i ng m i c r ogl i a l pr oduc t i on of pr o i nf l a m m a t or y m e di a t or s ( P a ha n, 1997; T r i nga l i 2004; N a t h, 2004) R e c e nt s t udi e s ha ve s how n t ha t l ova s t a t i n pos s e s s e s a nt i i nf l a m m a t or y a nd i m m unom odul a t or y p r ope r t i e s d i s t i nc t f r o m i t s a c t i on of l ow e r i ng s e r um l i pi d l e ve l s C ont r a s t i ng w i t h ot he r l i pi d l ow e r i ng s t r a t e gi e s l o va s t a t i n ha s s how n c l i ni c a l e f f i c a c y i n t he t r e a t m e nt of A l z he i m e r s d i s e a s e ( A D ) s ugge s t i ng t ha t l ova s t a t i n s t he r a pe ut i c e f f e c t i n A D m a y be de r i ve d f r om t he m odul a t i on of i m m une / i nf l a m m a t or y p r oc e s s e s G i ve n t he i m por t a nc e o f C D 40 a nd C D 154 i n ne ur ol ogi c di s e a s e s a nd t he c ont r i but i on of m a c r opha ge s / m i c r ogl i a t o t he di s e a s e pr oc e s s w e ha ve be e n pa r t i c ul a r l y i nt e r e s t e d i n t he r e gul a t i on of C D 40 e xpr e s s i on i n t he s e c e l l s I t ha s be e n r e por t e d t ha t I F N i s a pot e nt s t i m ul a t or of m i c r ogl i a l C D 40 e x pr e s s i on ( N guye n, 1998) I n a ddi t i on, w e ha ve pr e vi ous l y m a de t he obs e r va t i on t ha t t he A l z he i m e r s A pe pt i de c a n s yne r gi z e w i t h t he I F N s i gna l i ng pa t hw a y t o dr a s t i c a l l y i nd uc e m i c r ogl i a l C D 40 e xpr e s s i on a nd s ubs e que nt ne ur ot oxi c i t y ( T a n, 1999 a ) B a s e d on t hi s f i ndi ng, w e ha ve done e xt e ns i ve w or k on t he r ol e o f m i c r ogl i a l C D 40 e xp r e s s i on i n A D pa t hoge ne s i s ( T ow n, 2001; T a n\n51 2002) I n A D br a i n C D 40 e xp r e s s i on ha s be e n de t e c t e d i n m i c r ogl i a a ggr e ga t e s a nd c o l oc a l i z e d w i t h A pos i t i ve s e ni l e pl a que s ( T ogo 2 000) A r e c e nt publ i c a t i on de m ons t r a t e d a bunda nt C D 154 pos i t i ve a s t r oc yt e s i n A D br a i n i n c l os e a ppos i t i on t o r e a c t i ve m i c r ogl i a a nd A pos i t i ve s e ni l e pl a que s ( C a l i nga s a n, 2002) s ugge s t i ng a s t r oc yt e s m a y be t he s our c e of C D 154 i n A D br a i n. T he r ol e of l ova s t a t i n on pr o m ot i ng m i c r ogl i a l pha goc yt i c f unc t i on i s o f g r e a t i nt e r e s t gi ve n t ha t m e c ha ni s m s t ha t d r i ve t he c l e a r a nc e of c e r e br a l a m yl oi d unde r l i e t he pr i nc i pl e be hi nd m a ny t he r a pe ut i c s t r a t e gi e s f o r A D I nt e r e s t i ngl y, one s t udy ha s r e c e nt l y a ddr e s s e d t he r ol e of l ova s t a t i n on A D t yp e pa t hol ogy i n t he T g2576 m ous e m ode l ( P a r k 2003 ) T he y f ound t ha t w hi l e a 3 w e e k t r e a t m e nt w i t h l ova s t a t i n r e duc e d pl a s m a c hol e s t e r ol l e ve l s i n bot h m a l e a nd f e m a l e T g2576 m i c e i t a c t ua l l y e nha nc e d c e r e br a l A pe pt i de a nd a m yl oi d pl a que i n t he f e m a l e T g2576 m i c e T he a ut ho r s s ugge s t t ha t l ow pl a s m a c hol e s t e r ol m i ght be a r i s k f a c t or f or A D i n f e m a l e s U nf or t una t e l y, t hi s s t udy doe s not e xa m i ne m i c r og l i a l a c t i va t i on s t a t us ( m or pho l ogi c a l or i m m unophe not ypi c ) or pr ovi de a ny bi ol ogi c a l i nde x of l ova s t a t i n s C N S a c c e s s i bi l i t y dur i ng t he 3 w e e k t r e a t m e nt r e gi m e n. T he s e pa r a m e t e r s a r e c r i t i c a l t o a n a s s e s s m e nt of l ova s t a t i n s i n v i v o e f f e c t s on m i c r ogl i a f unc t i on o r e f f e c t s on A D pa t hol ogy. I n a not he r s t udy us i ng t he P S A P P m ous e m ode l o f A D t he C N S pe r m e a bl e c ho l e s t e r o l l o w e r i ng dr ug B M 15. 766 w a s a b l e t o r e du c e c e r e br a l A p e p t i d e l e v e l s a nd a m y l o i d p l a qu e l o a d ( R e f o l o 2001 ) s ugge s t i ng t h a t l o v a s t a t i n s l i m i t e d C N S a c c e s s i bi l i t y m i gh t e xp l a i n t h e e f f e c t s obs e r v e d by P a r k e t a l I n s um m a r y, w e s how t ha t l ova s t a t i n de c r e a s e s C D 40 e xpr e s s i on i n c ul t ur e d m ous e m i c r ogl i a l c e l l s B y r e duc i ng m i c r ogl i a l C D 40 e xpr e s s i on a nd s i gna l i ng, s t a t i ns\n52 s uc h a s l ova s t a t i n m a y r e duc e i nf l a m m a t i on a s w e l l a s pr om ot e m i c r ogl i a l pha goc yt i c f unc t i on, f e a t ur e s be l i e ve d t o be i m por t a nt i n o t he r t he r a pe ut i c s t r a t e gi e s f or A D O ur f i ndi ngs no t onl y pr ovi de a n a ddi t i ona l e xpl a na t i on f or t he pr e vi ous l y unknow n non l i pi d e f f e c t s of l ova s t a t i n on m i c r ogl i a l c e l l s but t he y a l s o s ugge s t t he i r a ppl i c a t i on i n A D a nd ot he r ne ur oi nf l a m m a t or y di s e a s e s i n w hi c h m i c r og l i a l C D 40 i s i nvol ve d.\n53 C H A P T E R F I V E C D 40 S I G N A L I N G R E G U L A T E S I N N A T E A N D A D A P T I V E A C T I V A T I O N O F M I C R O G L I A I N R E S P O N S E T O A 5. 1 I n t r od u c t i on A l z he i m e r s di s e a s e ( A D ) i s a ne ur ode ge ne r a t i ve d i s e a s e w i t h pr i m a r y ne ur opa t hol ogi c a l ha l l m a r ks of i nt r a c e l l ul a r ne ur of i br i l l a r y t a ngl e s a nd e xt r a c e l l ul a r s e ni l e pl a que s c om pr i s e d pr i m a r i l y o f a m yl oi d p e pt i de ( A ) A ddi t i ona l l y t he A D br a i n i s a l s o c ha r a c t e r i z e d by m a r ke d m i c r ogl i a m e di a t e d i m m une r e s pons e s t he r ol e of w hi c h ha s be c om e pot e nt i a l l y i m po r t a nt ye t unc l e a r O n t he one ha nd, a c t i va t e d m i c r ogl i a l c e l l s ha ve be e n i m p l i c a t e d i n A D pa t ho ge ne s i s by pr om ot i ng A pl a que f or m a t i on a s w e l l a s pr o i n f l a m m a t or y c yt oki ne pr oduc t i on ( W e gi e l 2000; W i s ni e w s ki 1994) ; on t he ot he r ha nd, s t udi e s ha ve s how n t ha t m i c r ogl i a l c e l l s a r e i nvol ve d i n pha g oc yt i c A pl a que c l e a r a nc e ( R oge r s 2000; 20 02) M i c r ogl i a l c e l l s a s r e s i de nt m a c r opha ge s of t he br a i n f or m t he f i r s t l i ne of de f e ns e a ga i ns t i nva di ng pa t hoge ns a nd pr ovi de a ke y l i nk be t w e e n t he c e nt r a l ne r vous s ys t e m ( C N S ) a nd t he i m m une s ys t e m I n t h e nor m a l a dul t br a i n m i c r ogl i a l c e l l s a r e r e l a t i ve l y qui e s c e nt but i n r e s pons e t o C N S i nj ur y, t he y a c t i ve l y pha goc yt os e c e l l ul a r de br i s a n d dyi ng c e l l s ( K r e ut z be r g, 1996) T hi s pha goc yt i c upt a ke of a popt ot i c c e l l s by m i c r ogl i a l c e l l s r e s ul t s i n r e duc e d pr o i nf l a m m a t or y c yt oki ne pr oduc t i on a nd m i ni m i z e s da m a ge i n t he i n f l a m e d br a i n ( M a gnus 2002)\n54 I n bot h t he c l i ni c a l s yndr om e a nd i n a ni m a l m ode l s of A D t he r e i s i nc r e a s e d a c t i va t i on a nd r e c r ui t m e nt of m i c r ogl i a l c e l l s t o a r e a s of c e r e br a l a m yl oi dos i s bu t t he s e a c t i va t e d m i c r ogl i a f a i l t o c l e a r A de pos i t s i n A l z he i m e r s m i c e ( S t a l de r 1997) H ow e ve r t he e m pl oym e nt of t he r a pe ut i c s t r a t e gi e s t ha t boos t m i c r ogl i a l pha goc yt i c a c t i va t i on i s a bl e t o r e duc e c e r e br a l A l oa d i n m o us e m ode l s of A D T hi s i s e xe m pl i f i e d by a r e por t s how i ng t ha t t r a ns f or m i ng gr ow t h f a c t o r be t a ( T G F ) ove r e xpr e s s i on pr om ot e s pha goc yt i c c l e a r a nc e of c e r e br a l A ( W ys s C or a y, 2001) S i m i l a r l y m i c r og l i a l F c r e c e pt or m e di a t e d r e m ova l of A i s pr opos e d a s one m e c ha ni s m by w hi c h S c he nk a nd c ol l e a gue s pe r i phe r a l i m m uni z a t i on of t he P D A P P m ous e m ode l o f A D w i t h A 1 4 2 r e s ul t e d i n r e duc e d A pl a que bu r de n ( S c he nk, 19 99) I nt e r e s t i ngl y m a c r opha ge c ol ony s t i m ul a t i ng f a c t or ( M C S F ) ha s a l s o be e n s how n t o pr om ot e m i c r ogl i a l pha goc yt i c r e m ova l of A a nd t he os t e ope t r ot i c m i c e w hi c h l a c k f unc t i ona l M C S F de ve l op C N S f i br i l l a r pl a que s ( K a ku, 2003) T a ke n t oge t he r i t s e e m s t ha t m i c r ogl i a l pha goc yt i c a bi l i t y c a n be m obi l i z e d t o pr om ot e A r e m ova l but e ndo ge nous m e c ha ni s m s f a i l t o e f f e c t i ve l y i ni t i a t e t hi s pr oc e s s W e ha ve pr e vi ous l y i de nt i f i e d t ha t m i c r og l i a l C D 40 r e c e pt or ( C D 40) pot e nt i a t e s A pe pt i de i nduc e d t um or ne c r os i s f a c t or a l pha ( T N F # ) pr oduc t i on a nd s ubs e que nt i n v i v o s t udi e s de m ons t r a t e d t ha t b l oc ka ge of C D 40 s i gna l i ng [ e i t he r b y a nt i body de pl e t i on of t he l i ga nd f o r C D 40 ( C D 40L ) or c r os s i ng w i t h C D 40L / m i c e ] w a s a bl e t o r e duc e c e r e br a l A / a m yl oi d l oa d i n a n A D m ous e m ode l ( T a n, 1999; 2002 ) C D 40 i s a ~ 45 50 kD a c e l l s ur f a c e m ol e c ul e a nd a m e m be r o f t he T N F # / ne r ve gr ow t h f a c t or r e c e pt or s upe r f a m i l y ( va n K oot e n, 2 000) A va r i e t y of pr of e s s i ona l a nd non pr of e s s i ona l a nt i ge n pr e s e nt i ng c e l l s ( A P C ) e x pr e s s C D 40, i nc l udi ng de ndr i t i c c e l l s B c e l l s m onoc yt e s / t i s s ue m a c r opha ge s a nd m i c r ogl i a l c e l l s ( va n K oot e n 2000) I t s\n55 c ogna t e l i ga nd, C D 40L i s t yp i c a l l y f ound on t he s ur f a c e of a c t i va t e d T l ym phoc yt e s but i t i s a l s o e xpr e s s e d i n t he C N S by a s t r oc yt e s M or e ove r e xpr e s s i on of C D 40 a nd C D 40L ha s be e n f ound i n a nd a r ound A p l a que s i n A D br a i n ( T ogo 2000; C a l i nga s a n, 2002) E f f e c t s of C D 40 l i ga t i on on m a t ur a t i o n, a c t i va t i on, a nd s ur vi va l o f de ndr i t i c c e l l s s ugge s t s t ha t C D 40 C D 40L i nt e r a c t i on pl a ys a n i m por t a nt r ol e i n A P C m e di a t e d i m m une r e s pons i ve ne s s ( K i kuc hi 2000; M c L e l l a n, 2000 ) I nc r e a s i ng e vi de nc e i n A D m ous e m ode l s s ugge s t s t ha t t he r e a r e m ul t i pl e f o r m s of m i c r ogl i a l a c t i va t i on, s om e m a ni f e s t a t i ons of w hi c h m a y a c t t o pr om ot e A / a m yl oi d pa t hol ogy, w hi l e o t he r f or m s m a y a c t t o r e m ove t h i s pa t hol ogy. W e pr opos e t w o t ype s of a c t i va t e d m i c r ogl i a l c e l l s i n t he c ont e xt o f A s pe c i f i c a l l y, a pha goc yt i c phe not ype a nd a n A P C phe not ype P r om ot i on o f t he p ha goc yt i c p he not ype w oul d be a s s oc i a t e d w i t h a nt i i nf l a m m a t or y c yt oki ne pr oduc t i on a nd w oul d i nhi bi t A pl a que f o r m a t i on w hi l e t he A P C phe not ype gi ve n t he r a r e pr e s e nc e of T c e l l s i n t he A D b r a i n, w oul d e s s e nt i a l l y r e s ul t i n unc he c ke d pr oduc t i on of A P C a s s oc i a t e d pr o i nf l a m m a t o r y c yt oki ne s t ha t w oul d a c t t o e xa c e r ba t e A D l i ke pa t hol ogy. G i ve n t he i r p l e i ot r ophi c p r e s e nt a t i on onc e a c t i va t e d, w e s ugge s t t ha t m i c r ogl i a l phe not ype s a r e de t e r m i ne d i n pa r t by t he i r l oc a l br a i n m i l i e u T hus i n t he c ont e xt of t he e vol vi ng A pl a que s of A D w e hypot he s i z e t ha t e nga ge m e nt of m i c r ogl i a l C D 40 r e pr e s e nt s one m e c ha ni s m t o s hi f t a c t i va t e d m i c r ogl i a l r e s pons e a w a y f r om pha goc yt i c a nd t ow a r ds a nt i g e n pr e s e nt a t i on, t hus r e nde r i ng t he s e c e l l s i ne f f e c t i ve a t A r e m ova l T o t e s t t hi s hy pot he s i s w e ha ve e m pl oye d pr i m a r y m ur i ne m i c r ogl i a l c e l l s t o i nve s t i ga t e t he r ol e of C D 40 s i gna l i ng i n pha goc yt os i s a nt i ge n pr e s e nt a t i on, a nd c yt oki ne r e s pons e of m i c r ogl i a l c e l l s t o A\n56 5. 2 R e s u l t s 5. 2. 1 T h e C D 40 C D 40L i n t e r ac t i on r e t ar d s m i c r ogl i al p h agoc yt os i s an d c l e ar an c e of A 1 4 2 p e p t i d e M i c r ogl i a l pha goc yt os i s of A ha s be e n de e m e d a pr i nc i pl e m e c ha ni s m gove r ni ng t he r e m ova l of A f r om t he b r a i n pa r e nc hym a ( B a r d, 2000) a nd t he C D 40 C D 40L i nt e r a c t i on i s a c r i t i c a l m odul a t or of i m m une c e l l a c t i v a t i on. W e w i s he d t o e va l ua t e w he t he r C D 40 l i ga t i on c oul d i nhi bi t m i c r ogl i a l upt a ke of A T hus w e a dde d a ge d F I T C t a gge d A 1 4 2 t o pr i m a r y m u r i ne m i c r og l i a l c e l l s f or 15 m i n 30 m i n a nd 60 m i n i n t he a bs e nc e or pr e s e nc e of a nt i A a nt i body ( c l one B A M 10 a s a pos i t i ve c ont r ol ) i s ot ype c ont r ol I gG C D 40L or he a t i na c t i va t e d C D 40L A s a c ont r o l f o r non s pe c i f i c i nc or por a t i on o f A by m i c r ogl i a m i c r ogl i a l c e l l s w e r e i nc uba t e d a t 4 o C i n pa r a l l e l c e l l c ul t ur e pl a t e s unde r t he s a m e t r e a t m e nt c ondi t i ons de s c r i be d a bove C e l l s upe r na t a nt s a nd l ys a t e s w e r e a na l yz e d f or e xt r a c e l l ul a r ( F i gur e 13) a nd c e l l a s s oc i a t e d ( F i gur e 14 ) F I T C A us i ng a f l uor om e t e r A s s how n i n F i gur e 13a b, da t a i ndi c a t e t ha t t he C D 40 C D 40L i nt e r a c t i on r e s ul t s i n de c r e a s e d m i c r ogl i a l u pt a ke of A 1 4 2 pe pt i de C ons i s t e nt w i t h a pr e vi ous r e por t ( R oge r s 2002 ) t r e a t m e nt w i t h t he pos i t i ve c ont r ol ( a nt i A a nt i body) m a r ke dl y i nc r e a s e d m i c r ogl i a l up t a ke of A 1 4 2 pe pt i de a t 60 m i n. I t s houl d be no t e d t ha t t he r e i s not a l w a ys a di r e c t c or r e l a t i on be t w e e n m i c r ogl i a l pha goc yt os i s a nd r e m ova l of A ( W e bs t e r 2000a b) T he r e f or e w e w i s he d t o de t e r m i ne w he t he r r e t a r de d m i c r ogl i a l pha goc yt os i s of A f o l l ow i ng C D 40 l i ga t i on a l s o pr oduc e d de c r e a s e d r e m ova l of t he pe pt i de W e t r e a t e d p r i m a r y c ul t ur e d m i c r ogl i a l c e l l s w i t h a ge d A 1 4 2 i n t he pr e s e nc e of C D 40L or he a t i n a c t i va t e d C D 40L pr ot e i n f or 6 h M i c r ogl i a l c ul t ur e m e di a w e r e c ol l e c t e d a nd i m m u nopr e c i pi t a t e d w i t h a nt i A a nt i body,\n59 a nd da t a s how e d t ha t C D 40L p r ot e i n t r e a t m e nt pr e ve nt e d A r e m ova l f r om t he c ul t ur e m e di a r e l a t i ve t o he a t i na c t i va t e d c ont r ol ( F i gu r e 1 5 a a nd b) I n a ddi t i on, m i c r ogl i a l c e l l s w e r e e xt e ns i ve l y w a s he d a nd l ys e d f or W e s t e r n bl ot a na l ys i s of A a nd de ns i t om e t r y s how e d t ha t C D 40L t r e a t m e nt r e s ul t e d i n a s i x f ol d r e duc t i on i n t he a m ount of c e l l a s s oc i a t e d A ( F i gur e 16) T a ke n t oge t he r t he s e da t a s ugge s t t ha t C D 40 l i ga t i on r e t a r ds t he a bi l i t y o f m i c r og l i a l c e l l s t o r e m ove a ge d e x oge nous l y a dde d hum a n A 1 4 2 5. 2. 2 M i c r ogl i al s e c r e t e d A P C as s oc i at e d c yt ok i n e s T N F # I L 6 an d I L 1 ar e s i gn i f i c an t l y i n c r e as e d i n t h e p r e s e n c e of A f ol l ow i n g C D 40L l i gat i o n W e ha ve pr e vi ous l y s how n t ha t l i ga t i on of m i c r ogl i a l C D 40 l e a ds t o t he s yne r gi s t i c a c t i va t i on of m i c r ogl i a l c e l l s by A a s e vi de nc e d by T N F # r e l e a s e a nd bys t a nde r ne ur ona l c e l l i nj u r y ( T a n, 1999) I n a ddi t i on, i t i s w e l l know n t ha t t he C D 40 C D 40L i nt e r a c t i on i s c r i t i c a l f or pr om ot i on of A P C di f f e r e nt i a t i on of m onoc yt e s m a c r opha ge s a nd de ndr i t i c c e l l s a s e vi de nc e d b y pr oduc t i on of pr o i nf l a m m a t o r y c yt oki ne s s uc h a s T N F # I L 6, a nd I L 1 ( K i kuc hi 2000) T o i nve s t i ga t e w he t he r C D 40 l i ga t i on pr om ot e s m i c r ogl i a l A P C di f f e r e nt i a t i on i n t he pr e s e nc e of A 1 4 2 pe pt i de w e e xa m i ne d c yt oki ne pr of i l e s i n t he s upe r na t a nt s of m i c r ogl i a l c e l l s c o t r e a t e d w i t h C D 40L p r ot e i n a nd A 1 4 2 pe pt i de f or 48 h D a t a s how t ha t c o t r e a t m e nt of m i c r ogl i a w i t h C D 40L pr o t e i n a nd A 1 4 2 pe pt i de s yne r gi s t i c a l l y i nc r e a s e d m i c r ogl i a l p r od uc t i on of A P C a s s oc i a t e d T N F # I L 6 a nd I L 1 c yt oki ne s ( F i gur e 17 19)\n64 5. 2. 3 C D 40 s i gn al i n g as s oc i at e d T h 1, b u t n o t T h 2, c yt o k i n e s i n h i b i t m i c r ogl i al p h agoc yt os i s of A 1 4 2 I t i s be c om i ng i nc r e a s i ngl y s ubs t a nt i a t e d t ha t t he C D 40 C D 40L i nt e r a c t i on pr om ot e s T he l pe r t ype 1 ( T h1 ) c e l l di f f e r e nt i a t i on a s e vi de nc e d by i nc r e a s e d pr oduc t i on of pr o i nf l a m m a t or y c yt oki ne s s uc h a s T N F # by t he A P C a nd I F N by t he T h1 c e l l ( D ong, 2001) I n or de r t o e xa m i ne put a t i ve m odul a t i on of m i c r ogl i a l pha goc yt os i s of A by C D 40 s i gna l i ng a s s oc i a t e d T h1 c yt oki ne s m i c r ogl i a l c e l l s ( i n 24 w e l l t i s s ue c ul t ur e pl a t e s 1 x 10 5 c e l l s / w e l l ) w e r e t r e a t e d w i t h 300 n M of a ge d F I T C t a gge d A 1 4 2 i n t he pr e s e nc e or a bs e nc e of r e c om bi na nt T h1 c yt o ki ne s ( I F N a nd/ or T N F # ) or T he l pe r t ype 2 ( T h2) c yt oki ne s ( I L 4 a nd/ or I L 10 a s c ont r ol s f or T h1 c yt oki ne s ) O ne hour a f t e r t r e a t m e nt c e l l c ul t ur e s upe r na t a nt s w e r e c ol l e c t e d a nd c e l l l ys a t e s w e r e pr e pa r e d t o m e a s ur e A by f l uor om e t r y. I n pa r a l l e l e xpe r i m e n t s m i c r ogl i a l c e l l s w e r e t r e a t e d w i t h 3 M of a ge d hum a n s ynt he t i c A 1 4 2 i n t he p r e s e nc e or a bs e nc e of t he T h1 or T h2 c yt oki ne s m e nt i one d a bove a nd 1 hour a f t e r t r e a t m e nt c e l l l ys a t e s w e r e s ubj e c t e d t o W e s t e r n a na l ys i s us i ng m ous e m onoc l ona l a nt i body c l one B A M 10, w hi c h s pe c i f i c a l l y r e c ogni z e s hum a n A A s c ons i s t e nt l y s how n w he n a na l yz i ng bot h m i c r ogl i a l c ondi t i one d m e di a ( F i gu r e 20 ) a nd l ys a t e s ( F i gu r e 21) C D 40 s i gna l i ng a s s oc i a t e d T h1 c yt oki ne s i nhi bi t w he r e a s T h2 c yt oki ne s boos t m i c r ogl i a l upt a ke o f A 1 4 2 5. 2. 4 C D 40 l i gat i on p r om ot e s f or m at i on of m i c r ogl i al M H C I I A p e p t i d e c o m p l e x H a vi ng s how n t ha t bot h C D 40 l i ga t i on a nd T h1 c yt oki ne s r e t a r de d m i c r ogl i a l upt a ke a nd c l e a r a nc e of A pe pt i de w e w onde r e d w he t he r C D 40L m i ght s hi f t m i c r ogl i a l a c t i va t i on t ow a r ds a n A P C phe not ype M a j or hi s t oc om pa t i bi l i t y c om pl e x I I ( M H C I I ) pl a ys a\n68 c r i t i c a l r ol e i n l oa di ng a nd t r a ns por t i ng of e xt r a c e l l ul a r pa t hoge ns a nd t oxi ns ( a s i m m un oge ni c M H C I I pe pt i de c om pl e xe s ) t o t he A P C s ur f a c e w he r e t he y a r e r e c ogni z e d by C D 4+ T c e l l s ( C how 2002) M H C I I i s pa r t i c u l a r l y i nt e r e s t i ng, a s m i c r ogl i a l c e l l s e xpr e s s i t i n t he f r ont a l c or t e x a nd h i ppoc a m pus of nor m a l a gi ng i ndi vi dua l s a nd e xpr e s s i on l e ve l s a r e m a r ke dl y i nc r e a s e d i n t he s e b r a i n r e gi ons i n A D c a s e s ( P e r l m ut t e r 1992) T he i m pa i r m e nt of M H C I I f unc t i on r e s ul t s i n a s i gni f i c a nt r e duc t i on of m i c r ogl i a a s s oc i a t e d C N S i nf l a m m a t i on ( i nc l udi ng m ul t i pl e s c l e r os i s a nd A D ) ( P e r l m ut t e r 1992; L i e w 1994 ) s ugge s t i ng t ha t M H C I I pl a ys a n i m por t a nt r ol e i n r e gul a t i on of m i c r ogl i a l i m m une r e s pons e s I n a dd i t i on, e f f e c t s of C D 40 l i ga t i on on pr om ot i on o f t he m i c r ogl i a l M H C I I m e di a t e d A P C pr oc e s s e s ha ve be e n a l s o r e por t e d ( M a t ys z a k, 1999; A l oi s i 2000 ; O ke e f e 2002) W e w i s he d t o i nve s t i ga t e w he t he r t he C D 40 C D 40L i nt e r a c t i on c oul d pr om ot e f or m a t i o n of i m m unoge ni c M H C I I A pe pt i de c om pl e xe s i n c ul t ur e d m i c r og l i a l c e l l s W e t r e a t e d m i c r ogl i a l c e l l s w i t h C D 40L pr ot e i n ( 2 g/ m L ) t he pr e s e nc e or a b s e nc e of a ge d P E A 1 4 2 pe pt i de ( 50 nM ) f or 60 m i n ( i n pi l ot s t udi e s w e e va l ua t e d va r i ous t i m e poi nt s f r o m 30 180 m i n, a nd f ound t ha t t he 60 m i n t i m e poi nt a l l ow e d opt i m a l c o l oc a l i z a t i on of M H C I I a nd A pe pt i de a s m e a s ur e d by i m m unopr e c i pi t a t i on/ W e s t e r n bl o t a nd f l uor e s c e nc e m i c r os c opy) ( da t a not s how n) T o e xa m i ne t he c o l oc a l i z a t i on of M H C I I a nd A pe pt i de w e pe r f or m e d i m m unof l uor e s c e nc e s t a i ni ng on t he s e c e l l s us i ng F I T C c onj uga t e d a nt i m ous e M H C I I a nt i body. R e s ul t s s how t ha t C D 40 l i ga t i on p r om ot e s M H C I I A pe pt i de c om pl e x f or m a t i on a s de t e c t e d by f l uor e s c e nc e m i c r os c opy ( F i gur e 22 a a nd b ) T o f ur t he r e va l ua t e t he s e e f f e c t s w e t r e a t e d pr i m a r y m i c r ogl i a l c e l l s i n pa r a l l e l w i t h C D 40L pr ot e i n ( 2 g/ m L ) i n t he pr e s e nc e or a bs e nc e of a ge d A 1 4 2 ( 3 M ) f or 60 m i n C e l l l ys a t e s\n69 w e r e t he n pr e pa r e d, i m m unop r e c i pi t a t e d w i t h a nt i m ous e M H C I I a nt i body, a nd s ubj e c t e d t o W e s t e r n bl ot t i ng us i ng a nt i hum a n A a nt i body ( B A M 10) A s s how n i n F i gur e 22 c da t a s how t ha t C D 40 l i ga t i on r e s ul t s i n pr om ot i on of M H C I I l oa de d A pe pt i de T a ke n t oge t he r t he s e da t a de m ons t r a t e t ha t C D 40 s i gna l i ng pl a ys a n i m por t a nt r o l e i n pr om ot i on of a M H C I I A c om pl e x. 5. 2. 5 M i c r ogl i al M H C I I A p e p t i d e c om p l e x i s f u n c t i on al I n or de r t o f ur t he r e va l ua t e t he f un c t i ona l i t y o f C D 40L s t i m ul a t e d M H C I I A pe pt i de c om pl e x, w e e s t a bl i s he d pr i m a r y c ul t ur e s of s pl e noc yt e s f r om C 57B L / 6 m i c e i m m uni z e d w i t h A 1 4 2 pe pt i de W e t he n i s ol a t e d C D 4+ T c e l l s f r om t he s e s pl e noc yt e s a nd pe r f or m e d a n a nt i ge n pr e s e nt a t i on a s s a y a s d e t a i l e d i n M a t e r i a l s a nd M e t hods B r i e f l y m i c r ogl i a l c e l l s w e r e p r e t r e a t e d w i t h A 1 4 2 ( 1 M ) i n t he p r e s e nc e or t he a bs e nc e of C D 40L pr ot e i n ( 2 g / m L ) f o r 6 h p r i or t o c o c ul t u r e w i t h A i m m un i z e d m ous e de r i ve d C D 4+ T c e l l s T hi r t y s i x hour s a f t e r c o c ul t ur e t h e r e s ul t i ng c o c ul t ur e d s upe r na t a nt s w e r e c ol l e c t e d f or a na l ys i s of c yt oki ne s A s s how n i n F i gu r e 23 l e ve l s of T c e l l de r i ve d c yt oki ne s ( i nc l udi ng I F N a nd I L 2 ) a nd m i c r ogl i a de r i ve d c yt oki ne s ( T N F # a nd I L 6 ) w e r e s i gni f i c a nt l y e l e va t e d i n t he c o t r e a t e d c ondi t i on ( C D 40L pl us A 1 4 2 ) c om pa r e d t o c ont r ol s de m ons t r a t i ng t ha t t hi s c ondi t i on s t i m ul a t e s bot h m i c r ogl i a l A P C f unc t i on a nd T c e l l a c t i va t i on F ur t he r m or e t o t e s t w he t he r t hi s e f f e c t c oul d be s pe c i f i c a l l y de pe nde nt on t he C D 40 C D 40L i n t e r a c t i on, w e pr e i nc uba t e d m i c r ogl i a l c e l l s w i t h a nt a goni s t a nt i C D 40 a nt i body ( 2. 5 g/ m L ) f or 1 h a nd r e pe a t e d t he a nt i ge n pr e s e nt a t i on a s s a y a s de s c r i be d a bove R e s ul t s s how t ha t s t i m ul a t i on o f m i c r ogl i a a nd T c e l l a c t i va t i on i s m a r ke dl y a t t e nua t e d a s e vi de nc e d by de c r e a s e d s e c r e t i on of I F N I L 2, T N F # a nd I L 6\n71 pr oduc t i on ( F i g. 23 a nd 24 ) s ugge s t i ng t ha t C D 40 s i gna l i ng i s ne c e s s a r y f or m i c r ogl i a l A P C phe not ype f unc t i on i n t he c ont e xt of A 5. 3 D i s c u s s i on W e ha ve pr e vi ous l y s how n t ha t i nt e r a c t i on of C D 40 w i t h C D 40L e na bl e s m i c r ogl i a l a c t i va t i on i n v i t r o i n r e s pons e t o A pe pt i de s ( T a n, 1999) T hi s f i nd i ng w a s va l i da t e d i n v i v o by t he obs e r va t i on o f de c r e a s e d m i c r ogl i os i s i n t r a ns ge ni c m i c e ove r pr oduc i ng A but de f i c i e nt i n C D 40L ( T a n, 2002) F ur t he r m or e r e duc e d m i c r ogl i os i s i n t he s e m i c e w a s a s s o c i a t e d w i t h m a r ke d r e duc t i on i n c e r e b r a l A l e ve l s a nd A pl a que l oa d. H ow e ve r not a l l f o r m s of m i c r ogl i a l a c t i va t i on a r e de l e t e r i ous a s a c t i va t e d m i c r ogl i a m a y s e r ve a pr ot e c t i ve r ol e i n i m m unot he r a pe ut i c s t r a t e gi e s f or A D ( R oge r s 2002; W ys s c or a y, 2001; S c he nk, 1999) H e r e w e s ugge s t a m ode l f o r m i c r ogl i a l a c t i va t i on ( F i gur e 25 ) t ha t a ddr e s s e s i t s s e e m i ngl y c ont r ove r s i a l r ol e a s bot h pot e nt i a l l y t he r a pe ut i c a s w e l l a s pa t ho ge ni c i n A D T hi s m ode l f i nds m a ny pa r a l l e l s w i t h t he a c t i va t i on of i nna t e i m m une c e l l s ( i e m a c r opha ge s a nd de ndr i t i c c e l l s ) i n t he pe r i phe r y a nd i s f u r t he r s uppor t e d by num e r ous s t udi e s t ha t ha ve a l r e a dy d e s c r i be d t he pl a s t i c i t y of C N S m i c r ogl i a l c e l l s a s w e l l a s t he i r pl e i ot r oph i c pr e s e nt a t i on onc e a c t i va t e d ( S a nt a m b r ogi o, 2001; N a ka m ur a 2002 ) O u r m ode l s ugge s t s t ha t m i c r ogl i a l c e l l s e xi s t i n a t l e a s t t w o f unc t i ona l l y di s c e r na bl e s t a t e s onc e a c t i va t e d na m e l y a pha goc yt i c phe not ype o r a n a nt i ge n p r e s e nt i ng phe not ype a s gove r ne d by t he i r c o s t i m ul a t or y e nvi r onm e nt I n t he pha goc yt i c phe not ype m i c r ogl i a l f unc t i on t o e ngu l f a nd de gr a de e xt r a c e l l ul a r de br i s or f or e i gn bodi e s W e s ugge s t t ha t t h i s pr ope r t y of t h e pha goc yt i c phe not ype i s a s s oc i a t e d w i t h t he r e po r t e d be ne f i t s o f a c t i va t e d m i c r ogl i a l c e l l s i n A D C onve r s e l y, s w i t c hi ng t o\n75 t he A P C phe not ype bl oc ks t hi s pha goc yt i c a bi l i t y a nd e l i c i t s t he r e l e a s e of p r o i nf l a m m a t or y c yt o ki ne s l e a di ng t o c hr on i c m i c r ogl i os i s w hi c h m a y a c t t o e xa c e r ba t e A D l i ke pa t hol ogy ( F r a c kow i a k, 1992) W e ha ve pr e vi ous l y de m ons t r a t e d t ha t c o a c t i va t i on of t he C D 40 s i gna l i ng pa t hw a y pot e nt i a t e s A s t i m ul a t i on of m i c r ogl i a l T N F # pr oduc t i on ( T a n, 1999 ) I n t hi s s t udy, w e e xt e nd on our e a r l i e r f i ndi ng by s how i ng t ha t t he C D 40 C D 40L i nt e r a c t i on c a n dyna m i c a l l y gove r n t he s w i t c h f r om t he pha goc yt i c t o t he a nt i ge n pr e s e nt i ng phe not ype of a c t i va t e d m i c r ogl i a l c e l l s i n v i t r o I n pa r t i c ul a r w e obs e r ve t ha t c o i nc uba t i on w i t h A a nd C D 40L pr ot e i n i nhi bi t s m i c r ogl i a l upt a ke a nd r e m ova l of e xoge nous A pe pt i de s w hi l e e voki ng t he r e l e a s e of T h1 t ype c yt oki ne s W e f ur t he r de m ons t r a t e t ha t t he s e T h1 c yt oki ne s c a n di r e c t l y i nhi bi t m i c r ogl i a l pha goc yt o s i s of e xoge nous A pe pt i de T hi s s ugge s t s a pos i t i ve f e e dba c k m e c ha ni s m w he r e by m i c r ogl i a l pr oduc t s o f t he A P C phe not ype dr i ve a c t i va t i on a w a y f r om a nt i i n f l a m m a t or y pha goc yt os i s a nd t ow a r ds pr o i nf l a m m a t or y a nt i ge n pr e s e nt a t i on. C ons i s t e nt w i t h t hi s vi e w w e r e por t i nc r e a s e d m i c r ogl i a l M H C I I A pe pt i de c om pl e xe s f ol l ow i ng C D 40 l i ga t i on i n t he p r e s e nc e of e xoge nous A pe pt i de T hi s s ugge s t s t ha t f ol l ow i ng C D 40 l i ga t i on, t he pha goc yt os e d A pe pt i de i s be i ng di r e c t e d a w a y f r om c l e a r a nc e m e c ha ni s m s ( i e l ys os om a l pa t hw a y) t ow a r ds M H C I I l oa di ng a nd pr e s e nt a t i on. M or e o ve r w e de m ons t r a t e t ha t t he s e M H C I I A pe pt i de c om pl e xe s a r e f unc t i ona l a s t he y m e di a t e T c e l l a c t i va t i on. T hi s m a y of be pa r t i c ul a r i m po r t a nc e i n i m m unot he r a pe ut i c s t r a t e gi e s f or A D a s pr e l i m i na r y e vi de nc e f r om t he r e c e nt l y ha l t e d A 1 4 2 va c c i na t i on c l i ni c a l t r a i l s s ugge s t t ha t A pe pt i de pr e s e nt a t i on by A P C s m a y ha ve c ont r i bu t e d t o t he i nva di ng T c e l l i nduc e d m e ni ngoe nc e pha l i t i s s e e n i n s om e o f t he pa t i e nt s ( F e r r e r 2004)\n76 I nt e r e s t i ngl y, be s i de s t he e s t a bl i s he d s i gna l i ng c a s c a de i ni t i a t e d by C D 40 l i ga t i on t he C D 40 r e c e pt or ha s be e n s how n t o di r e c t l y e ns ur e s ur f a c e bi ndi ng a nd upt a ke of he a t s hoc k pr ot e i n 70 ( H S P ) a s s oc i a t e d pr ot e i ns ( B e c ke r 2 002 ) I n f a c t H S P 70 a s s oc i a t e d pr ot e i ns ha ve be e n pr e vi ous l y s how n t o bi nd A p e pt i de s a nd f a c i l i t a t e t he i r pha goc yt os i s a nd c l e a r a nc e by m i c r ogl i a l c e l l s ( K a ki m ur a 2002 ) H ow e ve r a s w e ha ve not d i r e c t l y a s s e s s e d t he r ol e of e ndoge nous H S P a s s oc i a t e d pr ot e i ns i n t hi s s t udy, i t r e m a i ns t o be de t e r m i ne d t o w ha t e xt e nt t he y i m pa c t t he phe nom e non t ha t w e ha ve de s c r i be d he r e s pe c i f i c a l l y m i c r ogl i a l C D 40 s i gna l i ng i nd uc e d s w i t c hi ng f r om pha goc yt i c t o A P C phe not ype i n t he c ont e xt of e xoge nous A pe pt i de s I n s um m a r y w e s ugge s t t ha t s om e of c on f us i on a b out t he r o l e of m i c r ogl i a l a c t i va t i on, i n ge ne r a l m a y be r e m e di e d i f one c ons i de r s t ha t t he t e r m a c t i va t i on w he n i t i s a ppl i e d t o m i c r ogl i a l c e l l s pr oba bl y r e pr e s e nt s a t l e a s t t w o f unc t i ona l l y di s t i nc t s t a t e s M or e ove r w e pr ov i de da t a t ha t s ugge s t s C D 40 s i g na l i ng pa t hw a y s t i m ul a t i on a s one o f t he m e c ha ni s m s by w hi c h a c t i va t e d m i c r ogl i a l c e l l s a r e c onve r t e d f r om t he pha goc yt i c phe not ype t o t he a nt i ge n pr e s e nt i ng phe not ype W i t h r e s pe c t t o t he m i c r ogl i os i s oc c ur r i ng i n A D w e s ugge s t t ha t t he r a pe ut i c i nt e r ve nt i ons t ha t pr om ot e t he ph a goc yt i c phe not ype of a c t i va t e d m i c r ogl i a m i ght t hus a vo i d t he unw a nt e d s e que l a e of t he A P C phe not ype a nd s l ow pr ogr e s s i on of t he di s e a s e\n77 R E F E R E N C E S A l oi s i F ( 2001) I m m une f unc t i on o f m i c r ogl i a G l i a 36( 2) : 165 79. 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