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Divergent mechanisms utilized by SOCS3 to mediate IL-10 inhibition of TNF-a and nitric oxide production.. 2005

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DIVERGENT MECHANISMS UTILIZED B Y S0CS3 TO MEDIATE IL-10 INHIBITION OF TNF-a A N D NITRIC OXIDE PRODUCTION B Y MACROPHAGES b y POORAN QASIMI B . Sc . and B . A S c , S i m o n Fraser Un ive r s i t y , 2000 A THESIS SUBMITTED IN PARTIAL FULLFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE F A C U L T Y OF G R A D U A T E STUDIES EXPERIMENTAL MEDICINE THE UNIVERSITY OF BRITISH C O L U M B I A April 2005 © Pooran Q a s i m i , 2005 Abstract The cytokine , inter leukin-10 ( IL-10) , inhibi ts act ivat ion o f macrophages b y activators such as l ipopolysacchr ide ( L P S ) . H o w e v e r the mechan i sm b y w h i c h I L - 1 0 interferes w i t h L P S s igna l l ing is s t i l l unclear. One wel l -character ized s igna l l ing pa thway activated b y I L - 1 0 is that o f Stat3 (S ignal transducer and activator o f transcription), w h i c h is essential for the anti- prol iferat ive and ant i - inf lammatory actions o f I L - 1 0 o n macrophages. In our search for I L - 1 0 - induced, Stat3-regulated genes, we found a candidate be long ing to S O C S (suppressor o f cytokine s ignal l ing) f a m i l y o f negative regulators o f cy tokine s igna l l ing . U s i n g mutant I L - 1 0 receptor and dominant negative Stat3, w e show that I L - 1 0 induces S O C S 3 message and prote in i n a Stat3-dependent manner. H o w e v e r mere expression o f S O C S 3 protein i n macrophages was not sufficient to inhib i t T N F - a protein product ion i n response to L P S , suggesting that addi t ional IL -10 - induced signals are required. A n d indeed w e f ind I L - 1 0 s t imula t ion induces phosphoryla t ion o f tyrosine 204 o f S O C S 3 protein. In order to determine the role o f S O C S 3 i n I L - 1 0 inh ib i t ion o f macrophage act ivat ion, w e der ived ce l l l ines f rom S O C S 3 7 " and S O C S 3 + / " mice . SOCS3" 7 " macrophages respond to L P S i n a manner s imi la r to wi ld - type cel ls , but I L - 1 0 is less effective i n inh ib i t ing L P S - i n d u c e d T N F - a and N O product ion i n the SOCS3" 7 " cel ls as compared to S O C S 3 + / " macrophages. Reconst i tu t ion o f S O C S 3 7 " cel ls w i t h a wi ld - type S O C S 3 c D N A restored I L - 1 0 responsiveness. In order to determine w h i c h S O C S 3 doma in is important i n I L - 1 0 s igna l l ing , S O C S 3 7 " macrophages were reconstituted w i t h var ious S O C S 3 d o m a i n mutants. I L - 1 0 required a l l domains o f S O C S 3 protein for the inh ib i t ion o f T N F - a protein expression. H o w e v e r , for inh ib i t ion o f T N F - a m R N A expression, I L - 1 0 does not seem to require the K I R doma in o f S O C S 3 protein. In contrast, o n l y the two tyrosine residues, 204 and 221 , located i n the S O C S - b o x doma in are required for I L - 1 0 inh ib i t ion o f L P S - i n d u c e d i N O S i i protein expression and subsequent N O product ion. These studies demonstrate the importance o f S O C S 3 protein i n the ant i - inf lammatory act ion o f I L - 1 0 and that inh ib i t ion o f N O and T N F - a b y I L - 1 0 depends o n different domains o f S O C S 3 . Character izat ion o f S O C S 3 s igna l l ing domains and its immediate downstream targets w i l l a l l o w development o f therapeutic strategies, w h i c h replicate the benef ic ia l anti- inf lammatory act ion o f I L - 1 0 . i n TABLE OF CONTENTS , Abst rac t 1 1 Table o f Contents i v L i s t o f Tables v L i s t o f F igures v l L i s t o f Abbrev ia t ions V 1 1 Acknowledgemen t s x C H A P T E R 1: Introduction 1 1.1 Macrophages 1 1.2 L P S s igna l l ing 9 1.3 I L - 1 0 s igna l l ing 16 1.4 S O C S 3 protein 22 C H A P T E R 2: Mate r ia l s and Methods 32 C H A P T E R 3: Resul ts 39 C H A P T E R 4: D i s c u s s i o n 60 C H A P T E R 5: C o n c l u s i o n and Future Di rec t ions 73 B i b l i o g r a p h y 76 iv L I S T O F T A B L E S Tab le 1: Requirement for different S O C S 3 domains i n media t ing I L - 1 0 inh ib i t i on o f various macrophage responses. (Page 69) v L I S T O F F I G U R E S F igure 1: Schematic representation o f regulat ion o f i N O S protein b y L P S and I L - 1 0 i n macrophages. (Page 7) F igure 2: Schematic representation o f L P S s ignal l ing . (Page 12) F igure 3: Schematic representation o f I L - 1 0 s igna l l ing . (Page 19) F igure 4: The alternative names and domain structures o f var ious members o f the S O C S fami ly . (Page 23) F igure 5: Structure and mode l o f kinase inh ib i t ion b y J A B / S O C S 1 and C I S 3 / S O C S 3 . (Page 25) F igure 6: The S O C S - b o x targets proteins for proteasomal degradation b y several mechanisms. (Page 27) F igure 7: Induct ion o f S O C S 3 message b y I L - 1 0 i n a Stat3-dependent manner. (Page 40) F igure 8: Induct ion o f S O C S 3 protein b y I L - 1 0 . (Page 41) F igure 9: Ec top i c expression o f S O C S 3 protein is not sufficient i n i nh ib i t i on to comple te ly inhib i t T N F - a protein product ion i n response to L P S . (Page 43) F igure 10: I L - 1 0 induces phosphoryla t ion o f S O C S 3 protein at tyrosine 204 i n the S O C S - b o x domain . (Page 45) F igure 11: I L - 1 0 inh ib i t ion o f T N F - a protein expression requires S O C S 3 dur ing the early phase o f s igna l l ing . (Page 47) F igure 12: I L - 1 0 inhibi ts expression o f T N F - a m R N A i n a SOCS3-dependen t manner. (Page 48) F igure 13 A : Schematic representation o f var ious domain mutants. (Page 50) F igure 13B: Reconst i tu t ion o f S O C S 3 7 " cells w i t h W T and mutant S O C S 3 . (Page 51) F igure 14: I L - 1 0 requires a l l domains o f S O C S 3 protein for i nh ib i t i on o f T N F - a protein product ion. (Page 53) F igure 15: E x c l u d i n g K I R domain , I L - 1 0 requires a l l domains o f S O C S 3 protein for inh ib i t ion o f T N F - a m R N A expression. (Page 55) F igure 16: Y 2 0 4 and 221 o f S O C S 3 protein is important for i nh ib i t i on o f N O product ion b y I L - 1 0 . (Page 57) F igure 17: I L - 1 0 requires Y 2 0 4 / 2 2 1 o f S O C S 3 for i nh ib i t i on o f i N O S protein expression. (Page 59) F igure 18: Schematic representation o f the mechan i sm b y w h i c h I L - 1 0 m a y be inh ib i t ing L P S s igna l l ing pathway. (Page 70) v i L I S T O F A B B R E V I A T I O N S A A M O Al te rna t ive ly activated macrophage A k t V - A k t mur ine v i r a l oncogene h o m o l o g y 1 A P - 1 A c t i v a t o r Protein-1 A P C A n t i g e n presenting c e l l A T P Adenos ine triphosphate C a C a l c i u m C D 1 4 M o n o c y t e differentiation antigen C D 14 C D 8 0 / 8 6 Co-s t imula tory molecu le C D 8 0 / 8 6 c-Jun c-Jun mi togen activated kinase C A M O C l a s s i c a l l y activated macrophage C R E B c y c l i c A M P responsive element b i n d i n g protein D N A D e o x y r i b o n u c l e i c ac id E C L Enhanced Chemi luminescence E D T A Ethylene-diamine-tetraacetic ac id E L I S A E n z y m e - l i n k e d immunosorbent assay E R K Ext race l lu lar -s ignal regulated kinase F A C S Fluorescence-act ivated c e l l sor t ing/cel l -scanning G F P Green fluorescence protein G M - C S F Granulocyte macrophage co lony s t imulat ing factor G P 1 3 0 G l y c o p r o t e i n 130 ( IL-6 receptor) H O - 1 Heme-oxygenase-1 I C A M Intercellular adhesion molecu le IFN-A, Interferon-A, IKB IKB protein I K K IKB protein kinase v i i I L - 1 Interleukin-1 I L - 6 Interleukin-6 I L - 8 Interleukin-8 I L - 1 0 Interleukin-10 I L - 1 2 Interleukin-12 I L K Integr in- l inked kinase i N O S inducible ni t r ic ox ide J A K Janus kinase J N K c-Jun-N-terminal kinase K I R K i n a s e inh ib i to ry reg ion K O K n o c k - o u t L P S L ipopo lysacchar ide LJJF L e u k e m i a inh ib i to ry factor M O Macrophage M H C M a j o r h is tocompat ibal i ty complex M A P K M i t o g e n activated protein kinase M D - 2 M D - 2 protein M T P Macrophage inf lammatory protein M K K M A P K kinase M y D 8 8 M y e l o i d differentiation p r imary response gene 88 N F - K B N u c l e a r factor-KB N M D A N-mefhyl-D-aspartate N O N i t r i c ox ide p38 M A P K p38 mi togen activated protein kinase P B S Phosphate buffered saline P G E 2 Prostaglandin E 2 P I 3 - K Phosphot idy l inos i to l 3-kinase v i i i P K A Prote in kinase A P K C Prote in kinase C P M S F Phenylmethanosul fonly f luoride R N A R i b o n u c l e i c ac id R O O - React ive oxygen intermediates S A P K Stress activated protein kinase S D S S o d i u m dodecy l sulphate S H 2 Src h o m o l o g y 2 S O C S Suppressor o f cy tokine s igna l l ing S R Scavenger receptor S T A T S igna l transducer and activator o f t ranscript ion T C F / L E F - 1 T - c e l l / l y m p h o i d enhancer factor-1 T h r Threonine T I R Tol l - in ter leukin-1 receptor T I R A P T I R domain-conta in ing adaptor protein T L R 4 T o l l - l i k e receptor-4 T N F - a T u m o r necrosis factor-a T N F R T N F receptor T R A F 6 TNF- recep to r associated factor-6 T y r Tyros ine n g / m L Nanagrams per m i l l i l i t e r H g / m L M i c r o g r a m s per m i l l i l i t r e U / m L E n z y m a t i c unit per m i l l i l i t r e W T W i l d - t y p e Y 2 0 4 Tyros ine 204 residue Y 2 2 1 Tyros ine 221 residue Acknowledgements I w o u l d l ike to thank m y supervisor, D r . A l i c e M u i , for g i v i n g me the opportuni ty to be part o f her laboratory and gain this invaluable experience. I w o u l d l i ke to also thank m y supervisory committee, D r . V i n c e D u r o n i o , and D r . M i c h a e l C o x , for a l l their scientif ic advise, t ime and considerat ion w h i c h a l l owed me to beat deadlines. I w o u l d l ike to thank D r . B i l l Sah l and D r . Chr i s O n g for their support. Las t but not least, I w o u l d l ike to extend m y gratitude to m y colleagues i n D r . M u i ' s lab : A l i Ghanipour , A n d r e w M i n g L u m , Rup inde r D h e s i and Irfan M o l e d i n a . M y special thanks and gratitude goes to A n d r e w M i n g L u m , w h o helped me t remendoulsy w i t h a l l the northern data. A l s o , thanks to D r . A k i h i k o Y o s h i m u r a for p r o v i d i n g us w i t h the S O C S 3 domain mutants, and D r . N i c h o l a s Caca lano for p r o v i d i n g us w i t h the phospho-specif ic S O C S 3 antibodies. A s w e l l , thanks to the Transplant Trainee p rogram for p r o v i d i n g funding for m y project. T h i s experience w o u l d not have been the same without the support o f m y f a m i l y and friends, so a special thanks goes to them for their constant encouragment and faith i n me. x \ \ CHAPTER 1: Introduction 1.1 Macrophages: Functions The i m m u n e system is composed o f m a n y interdependent c e l l types that have specia l ized functions and co l l ec t ive ly protect the b o d y f rom bacterial , parasit ic, v i r a l infections and growth o f tumour cel ls . A m o n g these, the macrophage is one o f the most p le iot ropic , exh ib i t ing a broad range o f b i o l o g i c a l functions, i nc lud ing pro- and/or ant i - inf lammatory activit ies and phagocytosis [4]. T h e y also engul f and ingest foreign particles (innate i m m u n e response) and present these antigens to T-ce l l s (acquired immune response) and thus are often referred to as antigen-presenting cel ls ( A P C ) [5]. Th i s is an important first step i n the in i t ia t ion o f an acquired i m m u n e response, w h i c h they regulate through a var ie ty o f cytokines and co- st imulatory molecules [6]. Macrophage differentiation and act ivat ion states are greatly inf luenced b y environmental signals inc lud ing , m i c r o b i a l antigens and cytokines [5, 7, 8]. Thus the nature and consequence o f macrophage act ivat ion is different among different macrophage populat ions (e.g. bone marrow-der ived , human b l o o d monocytes , tissue macrophages f rom different sites, and macrophages isolated from inf lammatory lesions or wounds) and depends o n w h i c h cytokines are present [4]. 1.1.1 Macrophages: Types O v e r the past years distinct macrophage subsets have been characterized. These subsets inc lude c lass ica l ly activated macrophages ( C A M $ ) , al ternatively activated macrophages ( A A M $ ) , and Type-2 activated macrophages [9-11]. The best-studied are the C A M $ , w h i c h are induced b y pro- inf lammatory m i c r o b i a l molecules such as l ipopolysacchar ide ( L P S ) i n a T h i T - c e l l (cel l-mediated immune response) cytokine environment (e.g. Interferon-y ( fFN-y) , tumour 1 necrosis factor-a ( T N F - a ) ) and release inf lammatory and/or m i c r o b i c i d a l products [9, 12]. These cel ls are dis t inguished b y their ab i l i ty to produce ni tr ic ox ide ( N O ) i n addi t ion to their increased expression o f major h is tocompat ib i l i ty complex ( M H C ) class II and C D 8 6 , and their enhanced antigen-presenting capaci ty [13, 14]. B y increasing oxida t ive burst and N O release, C A M $ p l ay an important role i n protect ion against intracel lular pathogens [12]. T h e y also exert anti-proliferative and cy to tox ic activit ies, part ly due to their ab i l i ty to secrete N O and pro- inf lammatory cytokines ( T N F - a , interleukin-1 ( IL-1) , I L - 6 ) [9, 12, 15]. The observation that the development o f C A M $ is inhibi ted b y T h 2 T - c e l l cytokines , lead to characterizat ion o f alternatively activated macrophages ( A A M $ ) , w h i c h have a different phys io log i ca l function [16]. T h e y do not produce I L - 1 2 , but secrete I L - 1 0 and dr ive preferentially a T h 2 T - c e l l - l i k e (antibody-mediated) i m m u n e response [10, 11, 17]. T h e y also express arginase act ivi ty , an enzyme that competes w i t h inducib le n i t r ic ox ide synthase ( i N O S ) for L-a rg in ine [10]. Arg inase converts L-a rg in ine to urea and L-orn i th ine thus shunting the substrate away f rom the N O product ion pathway ( F i g . 1). has been shown to influence the differentiation o f T-ce l l s , and determine whether immuno-s t imula t ion versus i m m u n o - suppression or protective immune responses versus immuno-pa tho logy responses occur [12, 15, 18, 19]. Co-s t imula t ion o f L P S activated macrophages through the Fey receptor results i n inh ib i t ion o f I L - 1 2 synthesis and increased amounts o f I L - 1 0 as compared to L P S s t imulat ion alone [10, 20] . Furthermore, these cel ls swi tch f rom tr iggering a T h l T - c e l l response dominated b y I F N - y secretion, to the induc t ion o f a T h 2 T - c e l l response typi f ied b y I L - 4 secretion and increased ant ibody responses, m a i n l y o f the I g G l isotype [10, 21] . These cel ls are ca l led T y p e 2 2-activated macrophages. T h e y exhibi t functional s imilar i t ies to C A M $ and A A M ^ . L i k e they produce T N F - a , IL -1 and I L - 6 , but un l ike T y p e 2-activated macrophages do not produce I L - 1 2 , but secrete I L - 1 0 and dr ive preferential ly a T h 2 T - c e l l - l i k e immune response [10, 11, 17]. T h i s phenotype is s imi la r to w i t h the o n l y difference be ing that T y p e 2-activated cel ls do not express arginase ac t iv i ty [10]; suggesting that macrophage populat ions w i t h over lapping phenotypes and/or functions exist. Thus the c lass i f icat ion o f the dist inct macrophage populat ions is s t i l l operational due to absence o f def ini t ive molecula r markers. 1.1.2 Effectors and regulatory products of macrophages A c t i v a t i o n o f macrophages b y rFN-y or bacterial c e l l products such as L P S induces a number o f i m m u n o l o g i c responses such as product ion o f pro- inf lammatory mediators (e.g. I L - 1 , I L - 6 , T N F - a , chemokines , myeloperoxidase etc). Ac t i va t ed macrophages also induce m i c r o b i o c i d a l ac t iv i ty (e.g. release o f reactive oxygen and ni t rogen intermediates), lymphocyte act ivat ion (antigen processing and presentation) and tissue r emode l l ing (e.g. product ion o f elastase/collegenase/hyalruronidase enzymes) [11]. These responses are important for macrophages to exert their functions i n k i l l i n g bacteria, parasites, v i ra l - infected cel ls and tumour cel ls and p r o v i d i n g T - c e l l help. (i) Interleukin-1 (IL-1) I L - 1 is produced p r i m a r i l y b y activated macrophages, but can also be made b y other cel ls . IL -1 has a number o f phys io log ica l actions inc lud ing induc ing product ion o f tissue factor thus t r iggering the b lood-c lo t t ing cascade, decreasing b l o o d pressure and i nduc ing fever. B u t the predominant i m m u n e funct ion o f IL-1 is to enhance the act ivat ion o f T-ce l l s i n response to antigens and thus initiate an adaptive immune response [22]. There is an increase i n product ion 3 o f I L - 2 and its receptor b y T-ce l l s i n response to I L - 1 , w h i c h i n turn augments the act ivat ion o f the T-ce l l s i n an autocrine manner [22]. IL -1 also induces expression o f I F N - y b y T-ce l l s . T h i s effect on T - c e l l act ivat ion b y IL-1 is m i m i c k e d b y T N F - a w h i c h is another cytokine secreted b y activated macrophages, (i i) Interleukin-6 (IL-6) I L - 6 is a cy tokine i n the hematopoiet in f ami ly [23] produced b y activated macrophages, but also b y fibroblasts, endothelial cel ls and activated T-helper cel ls [24]. Its functions range f rom k e y roles i n acute-phase protein induc t ion to B and T - c e l l g rowth and differentiation [25, 26] . U n l i k e I L - 1 , I L - 2 and T N F - a , I L - 6 does not induce cy tokine expression, but rather augments the responses o f immune cel ls to other cytokines [27]. I L - 6 acts i n synergy w i t h IL -1 and T N F - a i n many immune responses, i nc lud ing T - c e l l act ivat ion [28]. I L - 6 induces transcript ion o f var ious proteins through the three major s ignal transduction pathways; protein kinase C , c A M P / p r o t e i n kinase A , and the c a l c i u m release pa thway [29]. I L - 6 stimulates the acute-phase reaction, w h i c h alerts the innate immune system and protects against tissue damage [30]. Th i s results i n the increases synthesis o f the two major acute-phase proteins, C-react ive protein ( C R P ) , w h i c h increases the rate o f phagocytosis o f bacteria, and serum a m y l o i d A ( S A A ) . S i m i l a r l y , it also increases the synthesis o f f ibr inogen, an important c lot t ing agent. The loca l acute phase react ion leads to a systemic reaction, w h i c h includes: fever, increased erythrocyte sedimentation rate, increased secretion o f g lucocor t icoids , and the act ivat ion o f the complement and clot t ing cascades. [31] Dysregu la t ion i n I L - 6 product ion has been shown to lead to septic shock and pathogenesis o f many diseases and autoimmune disorders, such as l iver autoimmune disease [32-34]. 4 ( i i i ) C h e m o k i n e s Chemok ines are among the most abundant proteins produced b y an activated macrophage. Interleukin-8 ( IL-8) , monocyte chemotactic protein-1 ( M C P - 1 ) and macrophage inf lammatory p r o t e i n - l a ( M l P - l a ) are the major leukocyte chemoattractants produced dur ing a bacterial infec t ion b y recrui t ing neutrophils, T-ce l l s and more macrophages to the site [35]. I L - 8 also stimulates neutrophils to degranulate. ( iv) T u m o r N e c r o s i s F a c t o r - a ( T N F - a ) T N F - a is a p le io t ropic inf lammatory cytokine produced b y several types o f cel ls , but p r i m a r i l y b y activated monocytes/macrophages [36]. It is an acute phase protein, w h i c h initiates a cascade o f cytokines and increases vascular permeabi l i ty , thereby recrui t ing macrophage and neutrophils to a site o f infect ion [37]. B i o l o g i c a l effects o f this molecu le inc lude induc t ion o f apoptosis, cy to lys is o f tumor cel ls , act ivat ion o f po lymorphonuc lear ( P M N ) leukocytes, ant ivi ra l ac t iv i ty and induc t ion o f I L - 1 [38, 39]. It possesses both growth s t imulat ing and growth inh ib i to ry properties. F o r instance, dur ing inf lammat ion , T N F - a induces neutrophi l prol i ferat ion, but upon b i n d i n g to the T N F R - 5 5 receptor it causes neutrophi l apoptosis [40]. S t imula ted macrophages produce membrane-bound 27 k d T N F - a , w h i c h can either b i n d d i rec t ly to T N F R - 5 5 and T N F R - 7 5 receptors through ce l l - to-ce l l contact, but the majori ty o f T N F - a prote in undergoes cleavage and binds i n its soluble form [41]. T N F - a secreted b y the macrophage causes product ion o f tissue factor result ing i n b l o o d c lo t t ing w h i c h serves to conta in the infect ion [42, 43] . In the absence o f T N F - a , m i c e infected w i t h gram-negative bacteria are susceptible to sepsis [44]. B u t h igh levels o f T N F - a correlate w i t h increased r i sk o f mor ta l i ty [37] and T N F - a also seems to be a central mediator i n various pathologies [43]. A few such examples include: septic shock, cancer, A I D S , transplantation rejection, mul t ip le 5 sclerosis, diabetes, rheumatoid arthritis, Crohn 's Disease, trauma, malar ia , meningi t is , i schemia- reperfusion injury, and adult respiratory distress syndrome. (v) Nitric Oxide (NO) Macrophages use the cyto toxic properties o f N O to el iminate parasites, bacteria and other potent ia l ly infectious particles [45]. In the vasculature, N O reacts w i t h i ron i n the active site o f the enzyme guany ly l cyclase ( G C ) , s t imulat ing it to produce the intracel lular mediator c y c l i c G M P ( c G M P ) that i n turn enhances the release o f neurotransmitters resul t ing i n smooth musc le re laxat ion and vasodi la t ion [45]. N O tox ic i ty is l i nked to its ab i l i ty to combine w i t h superoxide anions (02~) to fo rm peroxynitr i te ( O N O O ) , an o x i d i z i n g free radica l that can cause D N A fragmentation and l i p i d ox ida t ion [45]. N O is also a mediator i n inf lammatory diseases such as rheumat ism and arthritis. The enzyme, inducib le n i t r ic ox ide synthase ( i N O S ) , catalyzes the product ion o f N O f rom L-arg in ine (F ig . 1). W i t h i n macrophages, L-arginine can be metabol ized b y two different pathways that result i n the product ion of: (i) L-ci t rul l ine and N O b y i N O S ; and ( i i ) u reum and L-ornithine b y arginase. The regulat ion o f i N O S - a r g i n a s e balance b y T h l T - c e l l immune mediators ( I F N - 7 or L P S ) and T h 2 T - c e l l cytokines ( IL-4 and I L - 1 0 ) i n distinct macrophage populat ions reflects their po lar iza t ion to either c lass ica l ly or al ternatively activated macrophages. Studies have shown that l o w b l o o d pressure induced b y septic shock, as w e l l as in f lammat ion associated w i t h the development o f arthritis and k i d n e y disease is reversed w i t h L - N M M A (an i N O S inhibi tor) treatment [46]. Transgenic m i c e unable to generate N O dur ing the immune responses d isp lay a reduced inf lammatory response [47]. I L - 1 0 inhibi ts N O product ion b y either induc ing upregulat ion o f arginase expression, thus shunting away the L-a rg in ine substrate from the i N O S pathway, or b y inh ib i t ing expression o f i N O S protein itself. 6 Figure 1. Schematic representation of regulation of iNOS protein by LPS and IL-10 in macrophages. L P S activates N F K B pa thway w h i c h leads to induc t ion o f i N O S protein. i N O S protein uses L-a rg in ine as a substrate to synthesize ni t r ic ox ide and carl ine, o n the other hand the enzyme arginase, induced b y IL-10 , shunts the L-a rg in ine away f rom the i N O S pathway and degrades it into products such as L-orn i th ine and urea. I L - 1 0 can also inhibi t i N O S prote in direct ly, either b y inh ib i t ing its message or protein expression. v i ) Myeoldperoxidase (MPO) M P O , an i ron-conta ining protein, is found i n the azurophi l ic granules o f neutrophi l ic po lymorphonuc lear leukocytes ( P M N s ) and i n the lysosomes o f monocytes i n humans [11]. Its major role is to a id i n m i c r o b i a l k i l l i n g . M o n o c y t e s lose their M P O act iv i ty dur ing convers ion to tissue macrophages, therefore m i c r o b i c i d a l and cy to toxic ac t iv i ty o f macrophages is dependent m a i n l y on reactive o x y g e n intermediates ( R O I ) , N O and other substances w h i c h are s imi la r to those i n neutrophils [48-51]. Howeve r , macrophages m a y acquire M P O from their environment 7 (e.g. f rom ingested neutrophils) to participate i n their cy to tox ic mechanisms. M P O m a y have a role i n atherosclerosis, carcinogenesis, and degenerative neuro log ica l diseases. (v i i ) Prostanoids U p o n phagocytosis , macrophages release arachidonic ac id from esterified g lyce ro l phosphol ip ids o f the ce l l membranes [52, 53]. It is immedia te ly metabol ized into pro- inf lammatory agents ca l led prostanoids [especially prostaglandin E 2 ( P G E ) and pros tacyc l in (PGI ) ] . These factors induce vasodilatat ion, act synergis t ica l ly w i t h complement component C 5 a and L T B , mediate fever and m y a l g i a i n response to I L - 1 , i n the combina t ion w i t h b r a d y k i n i n and histamine they contribute to erythema, edema, and pa in induct ion . (v i i i ) Extracellular Proteases Macrophages not o n l y secrete cy to toxic and inf lammatory mediators, but they also release substances part ic ipat ing i n tissue reorganization, w h i c h include enzymes, such as hyaluronidase, elastase, and collagenase. Hyaluron idase reduces tissue v i scos i ty and a l lows for greater spreading o f mater ial i n tissue spaces b y destroying hya luron ic acid , an important component o f connect ive tissue. S i m i l a r l y , elastase and collagenase enzymes are i n v o l v e d i n degradation o f co l lagen and elastin, the basic components o f connect ive tissue, resul t ing i n disorganizat ion o f extracellular matr ix , w h i c h is important for the integrity o f the cel ls . 8 1.2 Macrophage Activation through LPS Signalling Pathway Lipopo lysacchar ide ( L P S ) is an integral component o f gram-negative bacterial c e l l w a l l , w h i c h el ici ts a broad spectrum o f b i o l o g i c a l activit ies [4]. L P S causes act ivat ion o f monocytes/macrophages, w h i c h are then able to recognize and k i l l foreign bacterial microorganisms [54]. L P S also induces product ion o f endogenous mediators such as T N F - a , I L - 1 , I L - 6 , I L - 8 , N O , superoxide anions, and l i p i d mediators [55, 56]. H o w e v e r , excessive amounts o f pro- inf lammatory cytokines m a y result i n fatal septic/endotoxic shock. W h e n cytokine p roduc t ion increases to such an extent that it escapes the l oca l infect ion, or when infect ion enters the bloodstream, sepsis develops [56, 57]. Systematic edema results i n l o w b l o o d vo lume , hypo-proteinanemia, neutropenia and then neutrophi l ia [44]. V i c t i m s o f septic shock experience fever, f a l l ing b l o o d pressure, myoca rd i a l suppression, dehydrat ion, acute renal failure, tissue damage and then respiratory arrest [42]. Tissue damage is brought on b y the loss o f b l o o d f low, w h i c h i n turn increases the product ion o f N O and leading to a further fa l l i n b l o o d pressure. Fa ta l i ty due to organ failure m a y occur. Therefore, i n order to understand h o w to main ta in regulated macrophage function dur ing inf lammatory responses, it is important to study the molecu la r mechanisms that both underl ie and l i m i t L P S s igna l l ing . 1.2.1 LPS Signalling L P S consists o f four regions that are structurally and funct ional ly distinct: the O - specific chain , the outer core, the inner core and the l i p i d A moie ty [4]. The conserved l i p i d A reg ion is responsible for the majori ty o f b i o l o g i c a l activit ies o f L P S [58]. The three molecules expressed o n the surface o f macrophages that are k n o w n to b i n d to l i p i d A moie ty o f L P S include: C D 1 4 , the macrophage scavenger receptor ( S R ) , and the [32 ( C D 1 1 / C D 1 8 ) leukocyte 9 integrins [59]. H o w e v e r , the s igna l l ing receptor for L P S consists o f to l l - l i ke receptor-4 ( T L R 4 ) and two other extracellular subunits, the g lycosy lphospha t idy l inos i to l - l inked C D 14 and soluble M D 2 protein, w h i c h increases the affinity o f L P S b i n d i n g [60-63] (F ig . 2). The serum protein, L P S b i n d i n g prote in ( L B P ) , accelerates the h igh affinity b i n d i n g o f L P S to C D 14 because it ca ta ly t ica l ly transfers L P S monomers from aggregates ( inc lud ing bacterial membranes) to C D 14 [64-66]. The importance o f C D 14 was shown b y experiments us ing b l o c k i n g monoc lona l antibodies to C D 1 4 result ing i n inh ib i t ion i n the abi l i ty o f L P S to stimulate phagocytes [59]. A soluble fragment o f C D 14 ( s C D 1 4 ) facilitates the act ivat ion o f cel ls that do not express membrane C D 14, such as endothelial cel ls [67-69]. M i c e deficient i n C D 14 are resistant to endotoxin shock after L P S challenge [70, 71]. It is general ly accepted that the interaction between l i p i d A and C D 14 is significant i n ce l lu lar act ivat ion o f L P S , but since C D 14 lacks a transmembrane and cy toplasmic domain , it must associate w i t h another transmembrane molecu le to transduce signals into the ce l l [57]. The transmembrane s igna l l ing subunit for the L P S receptor complex is T L R 4 and its d i scovery has greatly contributed to the understanding o f the molecu la r basis o f L P S recogni t ion and L P S - i n d u c e d s igna l l ing events [72]. The involvement o f T L R 4 i n L P S - i n d u c e d s igna l l ing events was demonstrated us ing LPS-hyporespons ive mice , w h i c h have either a point mutat ion i n T L R 4 [73, 74] or are T L R 4 - d e f i c i e n t [62]. A thi rd extracellular component o f the L P S receptor is M D - 2 . Exp re s s ion o f M D - 2 , as w e l l as T L R 4 , is required for reconsti tuting L P S responsiveness i n the non-macrophage, B a / F 3 , c e l l [75], and also human embryonic k i d n e y ( H E K ) 293 cel ls [76]. Down-regu la t ion o f ce l l surface expression o f T L R 4 / M D 2 complex i n mouse peri toneal macrophages correlates w i t h endotoxin tolerance, a state o f L P S unresponsiveness [77]. 10 Afte r L P S binds its receptor complex , T L R 4 associates w i t h the intracel lular adaptor proteins, M y d 8 8 and T I R A P proteins [22]. M y d 8 8 recruits the I R A K kinase w h i c h associates w i t h the adaptor prote in T R A F 6 , leading to act ivat ion o f the M A P 3 - k i n a s e s T a k l and M E K K 1 w h i c h phosphorylates and activates the IKB kinase ( I K K ) complex [78, 79]. I K K phosphoryla t ion o f IKB o n ser ine 2 3 targets IKB for recogni t ion and ubiqui t ina t ion b y the E 3 ub iqu i t in ligase complex , E 3 R S I k B [80] and degradation b y 26S proteasome. IKB regulates the act ivat ion o f the N u c l e a r factor K B ( N F K B ) , w h i c h is a t ranscript ion factor sequestered i n the cy top lasm b y IKB prote in [79]. IKB phosphoryla t ion b y I K K results i n release o f N F K B from the N F K B / I K B complex . T h i s phosphoryla t ion leads to the exposure o f the nuclear loca l iza t ion signals ( N L S ) on the N F K B subunits and the subsequent t ranslocat ion o f the molecu le to the nucleus [81]. 11 Figure 2. Schematic representation of LPS signalling. Afte r L P S binds its receptor complex , T L R 4 associates w i t h the intracellular adaptor proteins, M y d 8 8 and T I R A P proteins. M y d 8 8 recruits the I R A K kinase w h i c h associates w i t h the adaptor protein T R A F 6 , leading to act ivat ion o f the M A P 3 - k i n a s e s T a k l and M E K K 1 / 4 , P K R , c ra f and PI3-kinase. IKB phosphoryla t ion b y I K K results i n release o f N F K B from the N F K B / I K B complex . Th i s phosphoryla t ion leads to the exposure o f the nuclear loca l iza t ion signals ( N L S ) on the N F K B subunits and the subsequent translocation o f the molecule to the nucleus, where transcription o f regulated genes is induced. 12 1.2.2 Targets of L P S Signalling a) N F K B activation There are f ive N F K B subunit f ami ly members that form dimers: R e l A (p65), p50, R e l B , c - R e l , and p52 ; and they d imer ize i n various combinat ions w h i c h have differ ing D N A b i n d i n g affinity and transactivation potential [82]. The most c o m m o n and best-characterized form o f N F K B is the p65/p50 heterodimer, w h i c h binds to a consensus sequence (5 ' G G G A C T T T C C - 3 ' ) i n the promoter o f various genes and activates their transcription [82]. F o r instance, N F K B induces the transcript ion o f various interleukins (e.g. I L - 1 ) , cytokines (e.g. T N F - a ) and induc ib le ni t r ic ox ide synthase ( i N O S ) [83]. N F K B plays an important role i n the regulat ion o f i m m u n e responses, in f lammat ion , ce l l -cyc le progression, ce l l apoptosis, oncogenesis and var ious autoimmune diseases [84]. The act ivat ion o f N F K B is thought to be part o f a stress response as it is activated b y a variety o f s t imul i that inc lude T N F - a , I L - 1 , lymphokines , and U V [85]. P y r o l l i d i n e dithiocarbamate (an inhib i tor o f N F K B D N A b i n d i n g act ivi ty) comple te ly inhibi ts cy tokine product ion , showing that act ivat ion o f N F K B is essential for pro inf lammatory mediator product ion [39]. b) P K B activation L P S s t imulat ion o f protein kinase B ( P K B ) can also activate I K K complex b y phosphoryla t ion o f I K K at threonine 23 [86, 87]. A d d i t i o n a l l y , it has been shown that P K B stimulates the transactivation act ivi ty o f the R e l A / p 6 5 through induc t ion o f I K K and p38 M A P K [14]. Suppression o f I K K act ivi ty b y I L - 1 0 has been shown to lead to inact iva t ion o f L P S and T N F - a induced N F K B expression [88]. 13 c) ILK activation L P S also activates in tegr in- l inked kinase ( I L K ) , an ankyrin-repeat conta in ing serine/threonine prote in kinase, whose act iv i ty is regulated b y ce l lu lar levels o f phosphatidylinositol-3,4,5-tr isphosphate (PIP3) i n a phosphat idyl inos i to l -3 ' -k inase ( P I 3 - K ) dependent manner [89]. I L K induces phosphoryla t ion and act ivat ion o f prote in kinase B ( P K B ) and I K B , leading to act ivat ion o f N F K B , w h i c h then activates i N O S [90]. Besides regulat ion o f N F K B i n response to L P S , I L K also regulates the ac t iv i ty o f t ranscript ion factors such as P- c a t e n i n e - T C F / L E F - 1 ( T - c e l l / l y m p h o i d enhancer factor), A P - 1 (adaptor protein-1) [91] and C R E B ( c A M P responsive element b i n d i n g protein) [92]. 1.2.3 Activation of mitogen-activated kinases (MAPKs) Other important events i n L P S s igna l l ing inc lude act ivat ion o f the M A P kinases: J N K [85], E R K 1 / 2 [93] and p38 M A P K [94]. The contr ibut ion o f these pathways to expression o f the prototypic pro inf lammatory cytokine T N F - a has been w e l l studied. (i) JNK T N F - a t ranscript ion is regulated b y the c -Jun-N- terminal kinase ( J N K ) . A c t i v a t i o n o f J N K is necessary for phosphoryla t ion o f act ivat ing transcription factor 2 ( A T F - 2 ) and c-Jun (c-Jun mi togen activated kinase) w h i c h complexes w i t h the transcript ion factor C R E B to support t ranscript ion o f T N F - a [85]. Func t iona l analysis o f T N F - a promoter, showed enhancer elements i n the region conta in ing C R E B and N F - K B sites to be required for op t imal transcript ion o f the T N F - a gene i n response to L P S [85]. B u t concerted par t ic ipat ion o f c-Jun complexes and p50/p65 are required for the m a x i m a l L P S induc t ion o f the T N F - a promoter [85]. 14 ) E R K 1 / 2 L P S activates E R K through the serine/threonine kinase, T p l 2 , and the E R K pathway is required for cy toplasmic transport o f T N F - a m R N A [93]. The 3'-untranslated reg ion ( U T R ) o f the T N F - a transcript contains a wel l -character ized type II A U - r i c h element ( A R E ) i n v o l v e d i n both translational control and m R N A stabil i ty [94, 95] . L P S - i n d u c e d T p l 2 / E R K - t r a n s d u c e d signals target the A U - r i c h element i n the 3 ' U T R o f the T N F - a R N A and controls its cy toplasmic m R N A transport [93]. T p ^ ' m i c e also demonstrate resistance to L P S / D - G a l a c t o s a m i n e - i n d u c e d shock due to post-transcriptional defect i n the induc t ion o f T N F - a b y L P S [93]. ( i i i ) p38 M A P K p38 M A P K regulates translation o f T N F - a prote in [94]. L P S act ivat ion o f p38 M A P K is essential for r emov ing the T N F - a m R N A translational b l o c k and a l l o w i n g association o f T N F a m R N A w i t h po lyr ibosomes , the site o f active protein translation [94]. T h i s regulat ion is dependent o n the presence o f the A R E i n the 3 ' U T R . 15 1.3 B i o l o g i c a l A c t i v i t i e s o f I L - 1 0 A s discussed previous ly , the persistence o f inf lammatory processes often results i n tissue damage, or even fatality; result ing i n disorders such as: rheumatoid arthritis, C r o h n ' s disease, and septic shock [96]. Thus the immune system is faced w i t h a permanent challenge: h o w to control infect ion w h i l e l i m i t i n g tissue damage. Hence , ant i - inf lammatory mechanisms are mandatory for host su rv iva l . A m o n g the factors that l i m i t inf lammatory responses, the anti- inf lammatory cytokine , I L - 1 0 , is one o f the most important. I L - 1 0 is a k e y regulator o f both innate and acquired immuni ty . P roduced b y activated B - c e l l s , keratinocytes, monocytes and macrophages, I L - 1 0 was in i t i a l l y detected as a T h 2 T - c e l l product that inhib i ted the prol i ferat ion, development and function o f T h l T-ce l l s . The molecu la r c l o n i n g o f I L - 1 0 and subsequent studies u t i l i z i n g recombinant cytokine revealed that al though I L - 1 0 exerted direct effects on T-ce l l s [97, 98], its major site o f action was the activated macrophage [99]. A c t i v a t i o n o f macrophages b y I F N - y or L P S induces a number o f i m m u n e responses, many o f w h i c h are inhib i ted b y I L - 1 0 [100]. I L - 1 0 suppresses product ion o f p ro inf lammatory mediators: cytokines , i nc lud ing T N F - a [101], IL -1 [102] I L - 6 ; and chemokines such as M I P - l a [103], M C P - l a n d I L - 8 [104]. It also inhibi ts phagocytosis [3] b y inh ib i t ing reactive oxygen and ni trogen intermediate product ion i n monocytes/macrophages and neutrophils [101]. In addi t ion to l i m i t i n g and terminat ing inf lammatory responses, I L - 1 0 also regulates growth and/or differentiation o f B cel ls , N K cel ls , cy to toxic and helper T-ce l l s , mast cel ls , granulocytes, dendri t ic cel ls , keratinocytes, and endothelial cel ls [100]. B y inh ib i t ing expression o f co- s t imulatory molecules such as C D 8 0 / 8 6 , M H C II and I C A M - 1 , I L - 1 0 makes macrophages a poor A P C [105-108]. I L - 1 0 also inhibi ts the product ion o f I L - 1 2 , w h i c h together w i t h the inh ib i t ion o f other co-s t imulatory molecules suppresses p r imary al loantigen-specif ic T - c e l l 16 responses [20, 109]. I L - 1 0 induces a l ong last ing state o f non-responsiveness (anergy) i n T - cel ls , w h i c h cannot be reversed b y I L - 2 or b y s t imulat ion w i t h a n t i - C D 3 and a n t i - C D 2 8 [110]. These inh ib i to ry effects o f I L - 1 0 are not due to non-specif ic , g loba l suppression o f ce l lu lar metabol i sm, since product ion o f the ant i - inf lammatory mediators such as I L - 1 R antagonist and soluble T N F - a receptor are enhanced b y I L - 1 0 . V a r i o u s an imal m o d e l studies have substantiated the in vivo importance o f an anti- inf lammatory role for I L - 1 0 . Targeted disrupt ion o f the I L - 1 0 gene results i n m ice characterized b y inf lammatory b o w e l disease [111] and exaggerated i m m u n e reactions w h e n chal lenged w i t h antigen or L P S [112]. Admin i s t r a t i on o f I L - 1 0 , on the other hand, ameliorates disease i n such models o f endotoxemia [113], transplantation [114] and au to immuni ty [115]. In humans, the presence o f elevated endogenous I L - 1 0 is a posi t ive prognostic var iable i n autoimmune disease [116] and al logeneic transplant patients [117]. I L - 1 0 has recently entered c l i n i c a l trials for the treatment o f human inf lammatory b o w e l disease [118]. 1.3.1 I L - 1 0 S i g n a l l i n g P a t h w a y s i n M a c r o p h a g e s A s shown i n F igure 3, the IL-1 OR consists o f at least two subunits [119-122]. B o t h be long to the type II cy tokine receptor super-family, w h i c h inc lude a l l the I F N receptor proteins [123] and both are necessary for I L - 1 0 s ignal transduction. The p r imary l igand-b ind ing component designated I L - l O R a , binds I L - 1 0 w i t h h igh aff ini ty and i n the presence o f I L - 1 0 associates w i t h an accessory subunit ca l led I L - l O R p ( C R F 2 - 4 ) . L i k e other members o f the class II cy tokine receptor f ami ly , both I L - l O R a and IL-10RP possess a juxtamembrane box 1 m o t i f important for interaction w i t h members o f the Janus f ami ly kinases ( J a k l / T y k 2 ) [123]. L i g a n d - induced hetero-dimerizat ion o f the receptor chains results i n act ivat ion o f I L - l O R a - b o u n d J a k l 17 and I L - l O R p - b o u n d T y k 2 . Ac t iva t ed Jak kinases phosphorylate I L - 1 0 R a o n two cy toplasmic tyrosine residues ( Y 4 2 7 / 4 7 7 o f m I L - l O R l ; Y 4 4 6 / 4 9 6 o f h I L - l O R l ) [123] and create d o c k i n g sites for latent cy toplasmic transcription factors o f the Stat f ami ly [124]. In the case o f the I L - 10R, Stat3 is recruited to these phosphotyrosyl residues [125] and becomes phosphorylated b y receptor-bound Jak kinases. S ta t l also becomes tyrosine phosphorylated, but not to the same extent as Stat3 and the mechan i sm o f receptor recruitment is not clear. U p o n phosphoryla t ion, Stat3 and S ta t l hetero- and/or homodimer ize , translocate into the nucleus, b i n d specific sequences i n the promoters o f target genes and stimulate transcript ion [125, 126]. Studies us ing mutant I L - 1 0 R 1 l ack ing the tyrosine necessary for Stat3 act ivat ion, dominant-interfering Stat3 and a c o u m e r m y c i n d imer izable Stat3, have shown that the Stat3 pa thway is essential for the anti-proliferative act ion o f I L - 1 0 on macrophage c e l l growth [127]. The absolute requirement o f Stat3 i n media t ing the ant i - inf lammatory effects o f I L - 1 0 has been demonstrated [128]. Stat3 pathway is also essential for the ab i l i ty o f I L - 1 0 to inhib i t L P S - induced product ion o f the proinf lammatory cy tokine T N F - a [129]. 1.3.2 Genes r egu l a t ed b y I L - 1 0 t h r o u g h Sta t3 p a t h w a y I L - 1 0 has been shown to induce expression o f certain genes i n a Stat3-dependent manner. These genes inc lude the ce l l cyc l e inhibi tors , p l 9 I N 1 C 4 d [130], heme-oxygenase-1 ( H O - 1) [131], nuclear protein B c l 3 [132] ,which is a member o f IKB protein f a m i l y harbor ing ankyr in repeat domains and suppressor o f cy tokine s igna l l ing 3 ( S O C S 3 ) , w h i c h w i l l be discussed i n more detail o n page 31 . 18 IL-10 Figure 3. Schematic representation of IL-10 signaling. I L - 1 0 receptor contains two subunits, a andp\ w h i c h interact w i t h members o f the Janus fami ly kinases ( J a k l / T y k 2 ) . L igand- induced heterodimerizat ion o f the receptor chains results i n act ivat ion o f J a k l and T y k 2 , w h i c h then phosphorylate I L - l O R a on two cy toplasmic tyrosine residues ( Y 4 2 7 / 4 7 7 o f m I L - l O R a ; Y 4 4 6 / 4 9 6 o f h I L - l O R a ) and create d o c k i n g sites for Stat3 transcription factor, w h i c h is recruited to these phosphotyrosyl residues and becomes phosphorylated b y receptor-bound Jak kinases. U p o n phosphoryla t ion, Stat3 homodimer izes , translocate into the nucleus, b i n d specific sequences i n the promoters o f target genes and stimulate transcription 19 1.3.3 Targets of IL-10 in Inhibition of LPS Signalling a) Inhibition of N F K B activation A l t h o u g h several studies have reported inh ib i to ry act ion o f I L - 1 0 o n either the N F K B [22, 78-80, 107, 133-135] or M A P kinase pathways [39], the mechanisms govern ing this process is s t i l l unclear. I L - 1 0 pretreatment inhibi ts L P S and T N F - a induced N F K B D N A - b i n d i n g b y spec i f ica l ly disrupt ing the p65/p50 heterodimer complex i n human P B M C [133] and mur ine macrophages [135]. T N F - a gene transcript ion is suppressed b y I L - 1 0 [136, 137], poss ib ly b y interfering w i t h act ivat ion o f N F K B [88, 133, 138]. In alveolar macrophages, I L - 1 0 stabil izes IKB protein b y de lay ing its L P S - m e d i a t e d degradation and resul t ing i n delayed nuclear translocation o f the p65 subunit [139]. T N F - a induced I L - 8 product ion is regulated through an NFicB-dependent mechan i sm [140], w h i c h is also inhib i ted b y I L - 1 0 through inh ib i t ion o f N F K B transcript ional ac t iv i ty [104]. Therefore, NFicB-dependent t ranscript ional regulat ion is a target for the ant i - inf lammatory actions o f I L - 1 0 [135]. H o w e v e r , i n contrast, other studies have demonstrated that I L - 1 0 is unable to suppress L P S - i n d u c e d act ivat ion o f N F K B D N A b i n d i n g i n human monocytes [141]. These conf l ic t ing results m a y be due to different c e l l types used i n the studies (human P B M C and mur ine macrophages versus human monocytes) . b) Inhibiting production of other pro-inflammatory mediators I L - 1 0 interferes w i t h L P S - i n d u c e d product ion o f pro- inf lammatory mediators at the leve l o f t ranscript ion or m R N A stabili ty, protein translation or protein s tabi l i ty [137-139, 142, 143]. Incubation o f alveolar macrophages w i t h I L - 1 0 results i n the steady state m R N A levels o f T N F - a , IL -1 a and I L - 1 [3 and inh ib i t ion o f their gene expression [139]. In mur ine macrophages, I L - 1 0 acts p r i m a r i l y through post-transcriptional mechanisms b y des tabi l iz ing T N F - a m R N A 20 [142, 144] or b y inh ib i t ing gene translation v i a suppressing the act ivat ion o f p38 M A P K [94]. O f part icular note, I L - 1 0 was recently shown to suppress T N F - a translation b y inh ib i t ing the associat ion o f T N F - a m R N A w i t h po lyr ibosomes i n a mechan i sm also dependent o n the presence o f an intact A R E i n the 3 ' - U T R [94]. Studies have also shown that some o f the intracel lular mechanisms b y w h i c h I L - 1 0 inhibi ts pro inf lammatory cy tokine product ion b y LPS-ac t iva t ed macrophages are dependent o n de novo protein synthesis [3, 104, 142]. F o r instance, treatment w i t h cyc lohex imide , w h i c h b locks protein synthesis, antagonized IL-10-dependent inh ib i t ion o f T N F - a , I L - l p and I L - 6 m R N A expression and cytokine release, suggesting an involvement o f n e w l y synthesized proteins i n I L - 10 act ion [88, 133, 142]. c) I n h i b i t i o n o f r ecep to r expres s ion S igna l l i ng pathways are also inhibi ted b y suppression o f receptor expression, thus the poss ib i l i t y that I L - 1 0 inhibi ts L P S s igna l l ing b y inh ib i t i ng expression o f T L R 4 has been examined i n monocytes and polymorphonuclear leukocytes, where I L - 1 0 has been shown to inhibi t upregulat ion o f T L R 4 m R N A i n response to L P S [145]. 21 1.4 S u p p r e s s o r o f C y t o k i n e S i g n a l l i n g 3 ( S O C S 3 ) In our search for I L - 1 0 induced, Stat3-regulated genes, w e found a candidate be long ing to the S O C S (Suppressors o f C y t o k i n e S igna l l ing) f ami ly o f negative regulators o f cy tokine s ignal ing ( F i g . 4). The S O C S proteins are a f ami ly o f Stat- inducible genes va r ious ly referred to as C I S (cytokine- inducib le S H 2 protein) [146-148], S O C S [149] or S S I (Stat-induced Stat- inhibi tor) [150, 151]. The first member o f S O C S , C I S 1 , was c loned as an early gene induced b y var ious cytokines , act ing v i a the conserved intracel lular reg ion o f their respective receptors. The encoded prote in had a doma in that differed i n amino ac id sequence f rom a l l the k n o w n S H 2 domains . C I S plays an important role i n inh ib i t ion o f erythropoiet in and I L - 3 receptor s igna l l ing [152]. The second member o f the f ami ly was c loned b y three independent research groups and the protein was g iven three different names: 1) E n d o et a l , isolated a single posi t ive clone f rom a c D N A l ibrary , expressing a Jak-b ind ing protein, J A B , capable o f interacting w i t h the Jak kinase doma in [147]; 2) N a k a et a l , d iscovered Stat3-induced Stat3 inhib i tor (SSI ) b y us ing monoc lona l antibodies against a sequence m o t i f found i n the S H 2 doma in o f Stat3 [151]; and 3) Starr et a l , d iscovered a protein capable o f suppressing cy tokine s ignal transduction, S O C S 1 [40]. S O C S 1 and S O C S 3 have been shown to be essential for proper regulat ion o f interferon-y and I L - 6 responses, respect ively [152 1999]. 22 SOCS member; CIS/CIS 1 SOCS1/IAB/SSI1 SOCS2/CIS2/SSI2 S0GS3/CIS3 /S $13 SOCS4 SOCS5/CIS6 SOCS6/CIS4 SOC37/CIS7 c c c 2Z: SOCS KIR SH2. B O X Figure 4. The alternate names and domain structures of various members of the SOCS family. The kinase inhibitory region (KIR) of SOCS 1 and SOCS3 is dashed. This figure is adapted from Larsen, L. et al, 2002 [2]. 23 1.4.1 C o n s e r v e d D o m a i n s o f S O C S P ro t e in s a) K I R a n d S H 2 d o m a i n s S O C S proteins are a f ami ly o f seven proteins that possess a C- te rmina l , 70 amino ac id reg ion o f h o m o l o g y referred to as the S O C S b o x as w e l l as an S H 2 d o m a i n [153] as shown i n F igure 4. H o w e v e r , S O C S 1 and S O C S 3 are different f rom the rest o f their f ami ly members since they not o n l y have no introns, but also possess a 12 amino ac id extended S H 2 doma in that constitute a kinase inh ib i to ry reg ion ( K I R ) [153]. S O C S proteins have also been found to inhib i t the catalytic ac t iv i ty o f Jak and non-Jak f ami ly kinases [154] and m a y act o n other types o f s igna l l ing molecules [155] through their K I R domains . S O C S 1 and S O C S 3 co-precipitate w i t h Jaks upon cytokine s t imulat ion, and are able to inhib i t the kinase ac t iv i ty o f these, al though w i t h differ ing affinity and kinet ics [147, 152]. F igure 5 shows a schematic representation o f the structure and m o d e l o f kinase inh ib i t ion b y S O C S 1 and S O C S 3 . S O C S 3 has weaker affinity for b i n d i n g to the Y 1 0 0 7 o f Jak2, res iding inside an act ivat ion loop o f the kinase domain , J H 1 [152]. It is suggested that the p r imary b ind ing for S O C S 3 m a y not be Jak, but other molecules w i t h i n the s igna l l ing cascade, such as the phosphorylated cy tokine receptor or Stat proteins [148]. S O C S 3 has been shown to interact through its S H 2 doma in w i t h phospho-tyrosine sites o n the receptors for the cy tokine that induced it. These include the receptors for I L - 6 ( Y 7 5 7 / Y 7 5 9 ) [156], i n su l in [157], erythropoietin ( Y 4 0 1 ) [158] and lept in ( Y 9 8 5 ) [159]. 24 A kinase inhibitory region (KIR) extended SH2 •ubdomain (ESS) CH domain (SOCS box) JAB/SOCS-1 F i g u r e 5. S t r u c t u r e a n d m o d e l o f k ina se i n h i b i t i o n b y J A B / S O C S 1 a n d C I S 3 / S O C S 3 . ( A ) Schematic m o d e l o f the functions o f J A B and C I S 3 domains . T h e b o l d h ighl ighted circles represent the essential amino acids i n the kinase inh ib i tory region ( K I R ) and the extended S H 2 subdomain. (B ) The mode l o f J H 1 activation and inh ib i t ion b y J A B / S O C S - 1. B i n d i n g o f J A B to the act ivat ion loop prevents the access o f substrates and/or A T P to the catalytic pocket T h i s figure is adapted from Y a s u k a w a , H . et a l , 2000 [1]. 25 b) SOCS-box domain The conservat ion o f the S O C S - b o x doma in i n a l l S O C S proteins indicates that it p lays an important role i n the phys io log i ca l act ion o f these proteins. A l l S O C S proteins have been found to associate through their S O C S - b o x w i t h E long ins B and C [153]. The E l o n g i n B C complex was i n i t i a l l y identif ied as components o f the m a m m a l i a n transcript ion factor SIII, w h i c h w h e n interacting w i t h a th i rd protein ( E l o n g i n A ) , can increase the overa l l e longat ion rate o f R N A polymerase II i n v i t ro [160]. E l o n g i n B is ub iqu i t in - l ike protein and E l o n g i n C is s imi l a r i n sequence to S k p l protein, w h i c h through protein-protein interaction regulates ce l lu lar processes, such as c e l l cyc le , t ranscription and development [161]. E l o n g i n A stabil izes the E l o n g i n B C complex and the m o t i f b y w h i c h E l o n g i n A recognizes E l o n g i n C has amino ac id s imi la r i ty to the S O C S - b o x . Th i s E long ins B C complex recruits C u l l i n - 2 (Cu l -2 ) , R b x l , and the E 2 ubiqui t in-conjugat ing enzyme and this forms the E 3 ubiqui t in- l igase [162]. The p r o x i m i t y o f this ub iqu i t in ligase to S O C S - r e c r u i t e d s igna l l ing molecules can result i n the latter's ubiqui t ina t ion and degradation [163, 164]. F igure 6 shows a schematic representation o f several mechanisms used b y S O C S - b o x to target proteins for degradation. The role o f E l o n g i n s B C i n S O C S function however is not ent irely clear. V a r i o u s reports suggest that associat ion o f S O C S protein w i t h E long ins B C serves to protect [162, 165] rather than accelerate proteasome-mediated degradation [160]. Interaction o f S O C S - b o x w i t h E l o n g i n B C complex has been shown to marked ly increase the stabil i ty o f the S O C S 1 protein and protect it f rom proteasomal degradation [162, 166]. A study b y Hanada et a l , has s h o w n that relative to S O C S 3 w i l d type controls, T-ce l l s expressing S O C S 3 protein w i t h a mutant S O C S - box domain , express s ignif icant ly lower levels o f S O C S 3 protein due to destabi l izat ion o f the 26 protein, w h i c h is reversed w i t h over-expression o f E l o n g i n B C [165]. T h i s d icho tomy reflects variable abil i t ies o f different S O C S / E l o n g i n s complexes to recruit an ubiqui t in- l igase act ivi ty. Figure 6. The SOCS-box targets proteins for proteasomal degradation by several mechanisms. ( A ) Af te r S O C S b ind ing to J A K , e longin B and C are recruited to the S O C S - b o x , and the ub iqu i t in -homology doma in on S O C S - b o x is recogninzed b y the proteasome. (B) E long ins B and C b i n d to the S O C S - b o x and recruit an E 3 ligase, transferring ubiqui t in to the S O C S protein and targeting the complex for proteasomal degradation. U B L , ubuqui t in l i ke protein. Th i s figure is adapted f rom Larsen, L . et a l , 2002 [2]. 27 1.4.2 Tyrosine Phosphorylation of SOCS3 Protein Jaks and receptor tyrosine kinases can phosphorylate S O C S 3 at two tyrosine residues, 204 and 221 , w i t h i n the conserved S O C S - b o x domains w h i c h then a l lows it to b i n d to the S H 2 doma in o f the Ras inhib i tor p i 20 R a s G A P leading to inh ib i t ion o f S T A T 5 act ivat ion but main ta in ing E R K act ivat ion [167, 168]. S ince S O C S proteins inhib i t g rowth factor responses; tyrosine phosphoryla t ion o f S O C S 3 can ensure c e l l su rv iva l and c e l l cyc l e progression (proliferation) through Ras pathway. Recent ly , the same group have shown that tyrosine phosphoryla t ion o f S O C S 3 at Y 2 0 4 / Y 2 2 1 a l lows it to interact w i t h adaptor proteins N c k - 1 and C r k - L , and thus modulate their downstream s ignal ing [174]. Ano the r study has shown that S O C S 3 tyrosine phosphoryla t ion regulates protein stabil i ty and E l o n g i n B C interaction [169]. Tyros ine phosphoryla t ion decreased S O C S 3 protein half- l i fe b y disrupt ing the interaction between S O C S 3 and E l o n g i n B C , w h i l e S O C S 3 / S O C S 1 ch imera (3/1/3), w h i c h bound m u c h more strongly to E l o n g i n B C , was s ignif icant ly more stable than wi ld - type S O C S 3 [169]. Thus a p h o s p h o - S O C S 3 protein m a y serve different functions depending o n the s t imu l i , t ime and loca t ion i n the ce l l . 1.4.3 Role of SOCS3 in Inflammatory Diseases In case o f inf lammatory diseases such as rheumatoid arthritis, ulcerat ive col i t i s and C r o h n ' s disease, S O C S 3 is h igh ly expressed i n affected tissues [165]. In patients w i t h C r o h n ' s disease, there is consti tutive act ivat ion o f Stat3 i n T-ce l l s f rom c o l o n mucosa due to inab i l i ty o f S O C S 3 to inhib i t I L - 6 induced phosphoryla t ion o f Stat3 [170]. The mechan i sm under ly ing the inab i l i ty o f S O C S 3 to inhibi t IL-6 - induced Stat3 act ivat ion i n these patients is not k n o w n . Transgenic m ice expressing a dominant interfering S O C S 3 prote in exhibi t a more potent Stat3 28 act ivat ion and a more severe col i t i s than compared to the wi ld - type littermates [165]. These results suggest that S O C S 3 acts as a negative feedback regulator i n inf lammatory diseases, and therefore S O C S 3 m a y be u t i l i zed b y I L - 1 0 to exert its ant i - inf lammatory actions. 29 The focus of this thesis Hypothesis: S O C S 3 mediates I L - 1 0 deact ivat ion o f macrophages b y targeting s igna l l ing proteins activated b y L P S . The overa l l objective o f this proposal is to define at a molecu la r l eve l the mechan i sm b y w h i c h IL -10 - induced s igna l l ing events interfere w i t h those o f macrophage activators and characterize the role they p l ay i n media t ing I L - 1 0 ' s ant i - inf lammatory action. The importance o f the Stat3 pa thway i n the ant i - inf lammatory act ion o f I L - 1 0 has been established and some o f the Stat3-regulated gene induc t ion events i n v o l v e d i n macrophage deact ivat ion b y I L - 1 0 has been ident i f ied [132]. P r e l im ina ry experiments examin ing the I L - 1 0 responsiveness o f various candidate genes showed that S O C S 3 , a member o f a f ami ly o f negative regulators o f cy tokine s igna l l ing , is regulated i n a Stat3-dependent manner and m a y be important i n media t ing I L - 1 0 ' s ant i - inf lammatory action. In most other cy tokine receptor systems, the Stat pathway interacts w i t h and is modula ted b y the act ion o f other pathways. P r e l im ina ry observations suggest that S O C S 3 m a y be i n v o l v e d i n the mechan i sm b y w h i c h I L - 1 0 antagonizes L P S act ion [3]. In this study, w e set out to examine the modula t ion o f L P S - i n d u c e d cy tokine product ion b y determining the stage at w h i c h cytokine product ion (i.e. transcription, translation) is affected b y S O C S 3 expression. M i c e disrupted for S O C S 3 gene are embryon ica l ly lethal due to impa i red placental development [22]. A n a l y s i s o f these m i c e showed that dur ing trophoblast giant c e l l differentiation, S O C S 3 n o r m a l l y functions to modulate l eukemia inhib i tory factor ( L I P ) s igna l l ing . S i m i l a r l y , tetraploid rescued S O C S 3 _ / ~ m i c e die w i t h i n 3 weeks o f b i r th because o f cardiac monocyte hypertrophy caused b y a loss o f S O C S3-mediated inh ib i t ion o f L I F and C T - 1 s igna l l ing [169]. Thus , i n 30 order to study the effects o f loss o f S O C S 3 protein on I L - 1 0 s igna l l ing , w e der ived macrophage c e l l l ines from S O C S 3 _ / " fetal l ive r cel ls [152]. W e have also investigated w h i c h doma in o f S O C S 3 protein is important for inh ib i t ion o f L P S s igna l l ing pa thway i n response to I L - 1 0 b y reconsti tuting S O C S 3 7 " macrophages w i t h W T and mutant S O C S 3 . Therefore, determining the point at w h i c h S O C S 3 interferes w i t h L P S s igna l l ing pathways and ident i fy ing the specific S O C S 3 region responsible for this action w i l l g ive insights into the mechan i sm b y w h i c h S O C S 3 mediates inh ib i t ion o f macrophage act ivat ion and define new targets for S O C S 3 action. 31 CHAPTER 2: Material/Methods 2.1 Reagents A l l reagents were obtained from S i g m a ( O a k v i l l e , Canada) unless otherwise indicated. T N F - a E L I S A ki ts were obtained from B D Pharminogen (Miss i s sauga , Canada) . An t ibod ie s to S O C S 3 protein and p h o s p h o - S O C S 3 protein (phospho T y r 204/221) were k i n d l y suppl ied b y D r . N i c h o l a s Caca lano ( U C L A ) . E r k ant ibody was from C e l l S igna l l i ng (Missassauga , Canada) and i N O S ant ibody was from Santa C r u z (Santa C r u z , U S A ) . I L - 1 0 was k i n d l y suppl ied b y D r . K e v i n M o o r e ( D N A X Research Institute). 2.2 Cell Culture J774.1 mur ine macrophage c e l l l ine ( A m e r i c a n T y p e Tissue Cul ture Co l l ec t ion ) were cultured i n D u b e l c c o ' s M o d i f i e d Eag le ' s M e d i u m ( D M E M ) supplemented w i t h 9% (v/v) fetal c a l f serum ( F C S ) o n tissue culture grade dishes. Macrophages were ce l l l ines also der ived from embryonic fetal l ive r hematopoetic progenitor cel ls to generate S O C S 3 + / " and S O C S 3 7 " immor ta l i zed macrophage cel ls ( k i n d l y p rov ided b y D r . James Ihle, St. Jude C h i l d r e n ' s hospi ta l , T N ) . These cel ls were g r o w n i n Iscoves M o d i f i e d D u b e l c c o ' s M e d i u m ( E V I D M ) supplemented w i t h 9 % (v/v) F C S and 5% C 1 2 7 condi t ioned med ia (contains C o l o n y - S t i m u l a t i n g Factor-1) . A l l ce l ls were g r o w n at 3 7 ° C and 5% CO2 i n a standard tissue culture incubator. 2.3 CaCh Competent cells E . c o l i cel ls , D H 5 a , were g r o w n i n 2 m l l i q u i d broth ( L B ) m e d i u m overnight i n a 3 7 ° C shaker. The next day, inoculated 100 m l L B - m e d i u m w i t h 1 m l o f overnight culture and grew un t i l OD600=0 .45 or a litt le less (usually takes about an hour, i f O D d i d not reach then checked every 10 minutes) . The culture was ch i l l ed i n a flask on ice for about 15-30 minutes. The centrifuge tubes were p rech i l l ed o n ice. The culture was spun d o w n c o l d at 4000 r p m (2000g) for 10 32 minutes and the pellet was resuspended i n 1/3 o f o r ig ina l v o l u m e w i t h 100 m M ice c o l d CaCi2 and incubated o n ice for 30 minutes. The tubes were spun c o l d at 3000 r p m (1500 g) for 10 minutes. The pellet was then resuspended i n 1/25 o f o r ig ina l v o l u m e w i t h ice c o l d 100 m M CaCl2/15% g lycero l . The bacterial culture was then incubated at 4 ° C for a few hours. Bac te r ia l cel ls were al iquoted into prechi l led tubes (200 ul/tube) and stored at - 8 0 ° C . ( M o l e c u l a r C l o n i n g M a n u a l , Shambrook et al) 2.4 E . Co l i Transformation The CaCi2 competent E . c o l i cel ls (DH5o;) were thawed o n ice and used at 5 0 u l per transformation. 3-5 u l o f p l a smid was added to the cel ls and they were kept o n ice for 1 hour. C e l l s were put i n heat bath set at 4 2 ° C for 2 minutes and then put o n ice for 2 minutes, p r ior to the addi t ion o f 2 m l o f pre-warmed L B ( 3 7 ° C ) and incubat ion at 3 7 ° C incubator for 1 hour to a l l ow the bacteria to recover. Tubes were then spun d o w n at 12000 r p m and the supernatant was aspirated off. The bacterial pellet was resuspended i n 50 u l o f L B . T h e n L B + A m p i c i l l i n (100 u g / m L ) agar plates were streaked w i t h 50 u l o f bacterial culture. Co lon i e s were p i c k e d w i t h autoclaved toothpicks to inoculate 4 m l o f L B + A m p i c i l l i n (100 u g / m L ) solut ion. The cultures were g r o w n i n a 3 7 ° C shaker overnight for approximate ly 16 hours. ( M o l e c u l a r C l o n i n g M a n u a l , Shambrook et al) 2.5 Plasmid D N A prep (miniprep) D N A was isolated us ing M i n i p r e p k i t f rom Qiagen ( M a r y l a n d , U S A ) . The bacterial so lu t ion was centrifuged i n eppendorf tubes at 14000 r p m for a few seconds. L B was aspirated o f f and bacterial pellet was re-suspended i n 200 u l P I buffer (25 m M Tr i s p H 8.0, 10 m M E D T A p H 8.0, R N a s e also added) b y m i x i n g up and down . T h e n 200 u l o f P 2 buffer (0.2 N N a O H , 1% S D S ) was added to lyse the bacteria. The tubes were m i x e d thoroughly and 350 u l o f N 3 buffer 33 (3 M K O A C , 3 M H O A C p H 4.8-5.5, ethanol was also added) was added. E a c h tube was immedia te ly m i x e d gently to a l l o w K + i o n precipitate S D S w i t h genomic D N A . The tubes were spun at 12000 r p m for 10 m i n and the supernatants ( -750 ul) were transferred onto co lumns w i t h co l lec t ion tubes. The co lumns were spun at 12000 r p m for 1 m i n and 750 u l o f 7 0 % ethanol was added to the co lumn . The columns were spun at 12000 r p m for 1 m i n and the f lu id i n the co l lec t ion tubes were poured out so that co lumns c o u l d be spun for another 1 m i n to ensure a l l the residual ethanol has been spun out o f the co lumns . The co lumns were transferred into eppendorf tubes and D N A was eluted from co lumns b y addi t ion o f 50 u l o f water. Af t e r 1 m i n , the co lumns were spun at 12000 r p m for 1 m i n . The D N A concentrat ion was based o n spectrophotometer measurements at 260 n m . 2.6 Retrovirus Infection M y c - t a g g e d S O C S 3 constructs i n p M X - I R E S - E G F P (provided b y D r . A k i h i k o Y o s h i m u r a ) were transfected into P l a t - E v i r a l packaging c e l l l ine , us ing l iposomal-media ted transfection. P l a t - E cel ls were plated at 3 X 1 0 6 cel ls per plate. The next day, 0 .9u l o f 40 m M l iposome were added to 200 p i o f autoclaved water i n one tube and 3 p g o f D N A to 200 p i o f water i n another tube. The two solutions were m i x e d and incubated @ R T for 25 minutes and 2 m l o f D M E M was added to the mix ture and added to the P l a t - E cel ls for 6 hours. T h e n 2 m l o f 9 % D M E M was added to rescue the cel ls from starvation. The next day, m e d i a was aspirated o f f the P l a t - E cel ls and 2 m l o f 9% I M D M supplemented w i t h 5% C 1 2 7 condi t ioned med ia was added to the cultures. The next day, v i rus-conta in ing supernatant was col lected, centrifuged twice for 10 m i n at 15000 r p m and added to the SOCS3" 7 " cel ls ( 2 X 1 0 6 SOCS3" 7 " cel ls i n 6 c m plates). Infected cel ls expressing G F P were detected w i t h a fluorescence microscope . G F P expression was observable 1 day after infect ion, but was more evident on the second day after infect ion. These 34 cel ls were sorted based o n G F P expression us ing a Fluorescence Ac t iva t ed C e l l Sorter. Af te r sort ing, these cel ls were g r o w n i n 9% I M D M supplemented w i t h 5% C 1 2 7 condi t ioned media . 2.7 Determination of protein expression by Western Blot Analysis Parental J774.1 , S O C S 3 + / " , and S O C S 3 7 " macrophages ( 5 X 1 0 6 ) were treated w i t h m I L - 1 0 (100 n g / m L ) , L P S (10 n g / m L ) , or I L - 1 0 + L P S at the concentrations indicated, washed w i t h c o l d phosphate-buffered saline ( P B S ) , and lysed i n 200 u l o f buffer (150 n M N a C l , 50 m M T r i s - H C l , 2 m M E D T A , I m M N a V 0 4 , I m M N a F and 1%NP40) supplemented w i t h Comple te Protease Inhibi tor C o c k t a i l (Roche , M o n t r e a l , Canada). C e l l lysates were col lec ted f o l l o w i n g centrifugation at 15000 r p m for 10 minutes to remove the nuc le i and the prote in concentrat ion was measured us ing B C A method. A l i q u o t s o f ce l l lysate conta ining 100 ug o f protein were b o i l e d i n I X sod ium dodecy l sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) sample buffer and then subjected to S D S - P A G E . Proteins were subsequently electro-transferred onto a P V D F membrane ( B i o R a d , Canada). Membranes were b l o c k e d w i t h Tris-buffered saline ( T B S ) 3 % B S A at r o o m temperature for 1 hr, then incubated w i t h the appropriate p r imary antibodies i n T B S - 3 % B S A overnight on a shaker. The membranes were then washed several t imes w i t h T B S - 0 . 0 5 % T w e e n buffer and incubated w i t h f luorescently-labeled ( A l e x o r F l o u r 680) secondary antibodies ( M o l e c u l a r Probes, Oregan, U S A ) for prote in detection us ing the L i c o r Odyssey system. T h e f o l l o w i n g antibodies were used at 1:1000 d i lu t ion : a n t i - S O C S 3 [168], phospho-specif ic antibodies to Y 2 0 4 / Y 2 2 1 on S O C S 3 [168], a n t i - i N O S and a n t i - E R K . 2.8 Determination of TNF-a protein levels by Enzyme-Linked Immuno-sorbent Assay (ELISA) S O C S 3 + / " and S O C S 3 7 " macrophages were plated at 2 X 1 0 5 c e l l / w e l l i n I M D M conta ining 9 % serum supplemented w i t h 5% C 1 2 7 condi t ioned med ia i n 24 w e l l tissue culture plates and 35 g r o w n overnight. The next day, the m e d i a was changed to D M E M conta ining 9 % serum and cel ls were st imulated w i t h L P S (100 n g / m L ) or L P S + m I L - 1 0 (10 n g / m L ) for 2 hours. The supernatants were col lec ted and analyzed for the presence o f T N F - a ! protein us ing m o u s e - T N F - a E L I S A ki t . The 96 w e l l E L I S A plates were coated overnight w i t h 50 p i o f T N F - a capture ant ibody di luted 1:250 i n coat ing buffer ( I m M NaHCC>3, 3 m M NaCC»3). The we l l s were washed 5 times w i t h P B S / t w e e n - 2 0 (0.05%) and incubated w i t h 100 p i o f assay diluent ( 1 0 % F C S i n 1 X P B S ) for 1 hour to b l o c k non-specif ic b ind ing . 100 o f samples were added to each w e l l and incubated for 2 hours at R T . The wel l s were washed 5 t imes and incubated w i t h 50 p i o f biot in-conjugated detection antibody (1:250) for 1 hour. The we l l s were washed 5 times and incubated w i t h 50 p i o f avidin-conjugated H R P enzyme (1:250) for 30 minutes. T h e n we l l s are washed 7 t imes (30 sec each) and f ina l ly incubated w i t h 100 p i o f substrate. The substrate used was O-Phenylenediamine tablet (S igma, St. L o u i s , U S A ) d i sso lved i n phosphate-citrate buffer at f inal concentration o f 0.4 m g m l , and activated b y addi t ion o f 0 .03% hydrogen peroxide. The E L I S A plates were developed i n the dark for 30 minutes. The react ion was stopped w i t h 100 p i o f 3 M Sulfur ic acid. U s i n g an E L I S A plate reader, the we l l s were read at 492 n m . The standard curve was obtained b y us ing T N F - a (100 u g / m L ) w i t h the top concentrat ion o f 2000 p g / m L . Tr ip l ica te samples were assayed so that statistics cou ld be appl ied to calculate p-values (p-value < 0.01 indicates s ignif icance). 2.9 Detection of Nitrite production Ni t r i t e product ion was measured b y the Greiss method [127]. S O C S 3 + / " and S O C S 3 " / _ macrophages were plated at 2 X 1 0 5 c e l l / m L i n I M D M conta ining 9 % serum supplemented w i t h 5% C I 2 7 condi t ioned m e d i a i n 24 w e l l tissue culture plates and g r o w n overnight. The next day, the m e d i a was changed to D M E M containing 9 % serum and cel ls were st imulated w i t h either 36 L P S ( lOOng m l ) or L P S + I L - 1 0 (10 n g / m L ) for 24 hours. The supernatants were col lected and analyzed for the presence o f nitrite us ing the Gre iss assay. Seventy-f ive p i o f the culture supernatant was combined w i t h 75 u l o f greiss reagent: 1% (v/v) sulfani lamide, 0 . 1 % (v/v) naphthylethylenediamine hydrochlor ide , 2 . 5 % (v/v) phosphor ic ac id , and water, and incubated at r o o m temperature for 5 m i n . Absorbance at 550 n m was measured b y an E L I S A plate reader. S o d i u m Ni t r i t e (1 m M ) was used to generate a standard curve w i t h top concentrat ion o f 250 n M . Tr ip l ica te samples were assayed so that statistics cou ld be appl ied to calculate p-values (p-value < 0.01 indicates s ignif icance). 2.10 Determination of TNF-a RNA expression by Northern Blot Analysis S O C S 3 + / " and S O C S 3 7 " cel ls were treated for 1-2 hours w i t h L P S (100 n g / m L ) or L P S + I L - 1 0 (10 n g / m L ) . C e l l s were harvested i n 1 m l o f T r i Z o l . C h l o r o f o r m (400 ul) was added, and then samples were shaken v igo rous ly for 30 seconds and incubated for 15 minutes at r o o m temperature ( R T ) . The samples were then centrifuged (14000 rpm) for 10 minutes (4 °C) and the top aqueous phase was transferred into R N a s e free 1.5 m l eppendorff tubes. The samples were vor texed after addi t ion o f 500 u l o f c o l d i sopropanol and incubated for 15 m i n ( R T ) and centrifuged (14000 rpm) to precipitate the R N A . The supernatant was aspirated o f f and 1 m l o f c o l d 7 5 % ethanol was added to wash the pellet. Samples were centrifuged at 11500 r p m for 10 m i n . The supernatant was aspirated o f f and the pellet was left at R T for 10 m i n to air dry. O n c e the pellet was dry, it was resuspended i n 10 u l o f RNase-free water. Formaldehyde-ge l load ing buffer (formaldehyde, formamide, M O P S , 5 0 % g lycero l , I m M E D T A , 0 .25% bromopheno l blue, . 2 5 % xylene cyano l F F ) was added and R N A denatured b y heating for 10 minutes at 5 5 ° C . Equiva len t amounts o f R N A were resolved b y electrophoresis o n 1% agarose gel conta in ing 3 7 % formaldehyde, blotted onto a n y l o n membrane, and cross- l inked b y exposure to U V light. 37 The membranes were then prehybridized, hybridized, and washed according to standard procedures (Molecular Cloning Manual, Shambrook et al). The TNF-a and G A P D H probes were radiolabeled with [a- P] dCTP by the random priming method. 2.11 Statistical Analysis Data are expressed as the mean + SE. JMPIN4 software program was used to perform One-way A N O V A analysis and p-values < 0.01 were considered significant. 38 C H A P T E R 3: Results 3.1 Induction of SOCS3 message by IL-10 in a Stat3-dependent manner. In a p re l iminary survey o f S O C S fami ly members , w e found that I L - 1 0 treatment o f J774.1 cel ls induces S O C S 3 m R N A expression. S O C S 3 m R N A is induced b y I L - 1 0 as ear ly as 30 m i n and this expression is sustained for as l ong as 10 hours post s t imulat ion ( F i g . 7a). In order to investigate the role o f the Stat pathway i n I L - 1 0 regulat ion o f S O C S 3 , a dominant inh ib i to ry form ( lack ing the C- te rmina l transactivation domain , AStat) was constructed and re t rovira l ly transduced into J774.1 cel ls . Express ion o f AStat3 inhib i ted the ab i l i ty o f I L - 1 0 to induce S O C S 3 m R N A (F ig . 7b). In contrast, A S t a t l d i d not inhib i t I L - 1 0 induc t ion o f S O C S 3 i n these cel ls ( F i g . 7e) indica t ing that S O C S 3 m R N A expression is Stat3-dependent. N e x t w e wanted to determine whether the two tyrosines ( Y 4 2 7 / 4 7 7 for m I L - l O R ; Y 4 4 6 / 4 9 6 for M L - 1 0 R ) i n the cy toplasmic doma in o f the I L - 1 0 receptor ( IL-1 OR) were required for S O C S 3 induct ion. W e expressed either the wi ld - type ( W T ) or h I L - l O R : T y r F F mutant ( l ack ing both Y 4 4 6 and Y 4 9 6 ) i n J774 cel ls . C e l l s were treated w i t h human I L - 1 0 i n the presence o f a b l o c k i n g anti- m l L - l O R ant ibody to prevent h I L - 1 0 s t imulat ion o f the endogenous mouse I L - 1 OR. A s shown i n F i g . 7c, al though the W T IL-1 OR s ignal ing was able to induce S O C S 3 expression, the tyrosine mutant was not (F ig . 7d). 39 SOCS3 GAPDH c 0.5 i 2 I 5 1 p m b . c. d. e. 0 0.5 1 2 5 10 hours Parental • * mm m m -mm wm. mm wm m m 1 ^ s ^ t e ^ ^ f e ^ t f M ^ MM w • €P I P Wt tfft- S B mm ASTAT3 ML-10R: WT ML-10R: Tyr ASTAT1 FF Figure 7. Induction of S O C S 3 message by IL-10 in a Stat3-dependent manner. Parental J774.1 cells or cells expressing a dominant Statl (AStatl), dominant negative Stat3 (AStat3), wild-type hIL-10 (hIL10R:WT) or tyr-null (hIL10R:TyrFF) were treated for the indicated times with LPS or LPS + IL-10. (Cell lines described in O'Farrell et al E M B O J 17:1006, 1998 [3]). The R N A blot was analyzed by Northern analysis for SOCS3 mRNA expression and G A P D H mRNA to confirm equal R N A loading in each sample. 40 3.2 Induction of SOCS3 protein by IL-10. We then determined whether IL-10 induces SOCS3 protein expression, since mRNA expression does not always correlate with protein levels. SOCS3 protein is known to be very unstable, so to prevent SOCS3 protein degradation via the 26S proteasome, J774.1 cells were pretreated with 3 nM of MG132 (a proteasome inhibitor) for 30 minutes. These cells then were stimulated with LPS+IL-10 or IL-10 at various times (Fig. 8). SOCS3 protein expression was observed in response to IL-10 by 1 hour (Fig. 8b). Interestingly, LPS+IL-10 treatment induces SOCS3 protein even more rapidly (Fig. 8c) compared to IL-10 alone. LPS alone was not able to induce SOCS3 protein at any time point (Fig. 8a). a. LPS Ctl 15 30 60 120 240 ( b. IL-10 ""*ftlj|*miii..i. : • i mm Ctl 15 30 60 120 240 (• C. LPS+IL-10 " Non-specific band SOCS-3 (33 kDa) Non-specific band SOCS-3 (33 kDa) • Non-specific band SOCS-3 (33 kDa) Ctl 15 30 60 120 240 (min) Figure 8. Induction of SOCS3 protein by IL-10. J774.1 cells were treated with control buffer or (a) 10 ng/mL LPS, (b) 100 ng/mL IL-10 or (c) 10 ng/mL LPS with 100 ng/mL IL-10 for the indicated length of time. Lysates were made IX in SDS-PAGE sample buffer, resolved by SDS-PAGE and subjected to immunoblot analysis with antibody to SOCS3 protein. The top bands are non-specific showing equivalent protein levels in each sample. These are results from 3 independent experiments. 41 3.3 Ectopic expression of SOCS3 protein is not sufficient to completely inhibit TNF-a production in response to LPS. Once it was determined that S O C S 3 m R N A was induced b y I L - 1 0 i n a Stat3-dependent manner, w e ec topica l ly expressed S O C S 3 protein i n J774.1 mur ine cel ls . B o t h const i tut ively expressing S O C S 3 and parental J774 cel ls were st imulated w i t h L P S + I L - 1 0 for 10 hours and their supernatants were col lec ted for T N F - a determination b y E L I S A . F igure 9 shows that J774.1 cel ls const i tut ively expressing S O C S 3 protein make less T N F - a protein i n response to L P S as compared to the control group. N o t a b l y however , complete inh ib i t ion o f T N F - a product ion required addi t ion o f I L - 1 0 . 42 20000 f g> 15000 + LPS + IL10 Parental SOCS 3 F i g u r e 9. E c t o p i c express ion o f SOCS3 p r o t e i n is not suff ic ient to comple t e ly i n h i b i t T N F - a p r o d u c t i o n i n response to L P S . W e ec topica l ly expressed S O C S 3 protein i n J774.1 murine cel ls . These cel ls were st imulated w i t h L P S + I L - 1 0 and their supernatant was tested for T N F - a levels after 10 hrs v i a an E L I S A . (This E L I S A assay was carried out b y D r . A l i c e M u i ) 43 3.4 IL-10 induces phosphorylation of SOCS3 protein at tyrosine 204 in the SOCS-box domain. Since mere expression o f S O C S 3 protein i n macrophages was not sufficient to inhib i t T N F - a : protein product ion i n response to L P S , this suggests that addi t ional I L - 1 0 signals are required. It has been shown that S O C S 3 protein is phosphorylated i n response to cytokines , g rowth factors and b y several famil ies o f kinases, i nc lud ing Jaks and receptor tyrosine kinases [168]. J774.1 cel ls were pretreated w i t h 10 u M N a V 0 3 and 3 u M M G 1 3 2 (a proteasome inhibi tor) for 30 minutes pr ior to s t imulat ion w i t h 100 n g / m L o f L P S + I L - 1 0 (100 n g / m L ) or I L - 10 alone. I L - 1 0 was able to induce phosphoryla t ion o f S O C S 3 protein at Y 2 0 4 (F ig . 10a) at 2 hours post s t imulat ion. The blot was re-probed for protein S O C S 3 ant ibody ( F i g . 10c), and later for E r k 1/2 protein ( F i g . lOd) to conf i rm equal protein loading. Phospho-speci f ic ant ibody raised against Y 2 0 4 was tested for its specif ici ty. The phospho-specif ic Y 2 0 4 ant ibody detected S O C S 3 protein i n cel ls expressing W T or S O C S 3 Y 2 2 1 F protein, but not i n S O C S 3 _ / " or Y204F S O C S 3 , Z U H r m a c r o p h a g e s (F ig . lOe). The same blot was reprobed w i t h a n t i - S O C S 3 ant ibody to con f i rm the presence o f S O C S 3 protein i n these samples ( F i g . lOg) . Ano the r blot was probed for p h o s p h o - Y 2 2 1 , and there were bands i n I L - 1 0 treated samples ( F i g . 10b). H o w e v e r , the phospho-specif ic Y 2 2 1 ant ibody d i d not show specif ic i ty (F ig . lOf) . 44 Q_ o + a. Ant i -PY204 b. An t i -PY221 C. Anti-SOCS3 d. Anti-Erkl/2 WT KO Y204F Y221F Anti-PY 204 Anti-PY 221 Anti-SOCS3 Figure 10. IL-10 induces phosphorylation of SOCS3 protein at tyrosine 204 in the SOCS-box domain. J774.1 cells were pretreated with 10 uM NaV03 and 5 uM MG132 proteosome inhibitor for 1 hour at 37°C. Then the samples were treated with control buffer or 100 ng/mL LPS + 100 ng/ml IL-10 or 100 ng/mL IL-10 alone for 2 hrs before preparation of cell lysates. Lysates were made I X in SDS-PAGE sample buffer, resolved by SDS-PAGE and subjected to immunoblot analysis with antibodies to phospho-tyr 204 SOCS3 (PY204), phospho-tyr 221 SOCS3 (PY221) or SOCS3 protein. The same blot was reprobed for Erk protein to confirm equal protein loading in each sample. SOCS 7" cells and SOCS"" reconstituted with cDNA for SOCS3 (WT), Y204F or Y221F mutants of SOCS3 were treated LPS+IL-10 for 2 hrs and samples were treated as above. The samples were subjected to immunoblot analysis with antibodies to PY204 (lOe) and PY221 (101). The blots were then reprobed with anti-SOCS3 antibody to confirm presence of SOCS3 protein (lOg). This experiment was carried out 3 independent times. 45 3 . 5 I L - 1 0 inhibition of T N F - a protein expression requires S O C S 3 during the early phase of signaling. T N F - a is a p r imary mediator o f numerous i m m u n o l o g i c functions, i nc lud ing in f lammat ion and regulat ion o f immune proliferat ive and act ivat ion responses. W e chose to study T N F - a as one o f the readouts for an activated macrophage due to f o l l o w i n g reasons: 1) T N F - a product ion is a ha l lmark characteristic o f macrophage act ivat ion, 2) T N F - a expression is regulated at mul t ip le levels and 3) S igna l transduction mechanisms i n v o l v e d i n this regulat ion are wel l -character ized. In order to determine the potential role o f S O C S 3 protein i n I L - 1 0 inh ib i t i on o f T N F - a prote in expression, we examined product ion o f T N F - a i n response to L P S + I L - 1 0 after 2 hours, i n the S O C S 3 + / " vs SOCS" 7 " macrophages. I L - 1 0 was able to inhib i t p roduct ion o f T N F - a i n S O C S + / " cel ls and not i n S O C S 3 " 7 " macrophages ( F i g . 11, panel A ) . T o conf i rm that the inh ib i t ion i n these cel ls are t ru ly due to the absence o f S O C S 3 protein, w e examined the product ion o f T N F - a i n S O C S3"7" macrophages reconstituted w i t h S O C S 3 c D N A v i a retroviral transduction. SOCS3" 7 " cel ls reconstituted w i t h S O C S 3 c D N A regained their responsiveness to I L - 1 0 and were able to inhib i t T N F - a product ion s imi la r to S O C S + / " macrophages (F ig . 11, panel A ) . B u t w h e n S O C S 3 + / " and S O C S 3 " 7 " cel ls were treated w i t h L P S + I L - 1 0 for 4 hours, I L - 1 0 was able to inhibi t T N F - a product ion both i n S O C S 3 + / " and S O C S 3 7 " cel ls (F ig . 11, panel B ) . These results suggest that the dependence o f I L - 1 0 o n S O C S 3 for i nh ib i t ion o f T N F - a product ion occurs o n l y dur ing the early phase o f I L - 1 0 s igna l l ing . 46 A. unstimulated LPS(100ng/mL) LPS+IL-10 (lOng/mL) SOCS3 SOCS3 SOCS3 -'- :WT LPS 362+4.9 350+6.0 372+4.2 S0CS3 SOCS37" SOCSS-^WT L+10 147+1.4 282+1.4 128+2.3 B. o y 3 C P. unstimulated LPS (100 ng/mL) LPS+IL-10 (lOng/mL) LPS L+10 S0CS3 + / " 1847+1.3 2 4 9 ± 1 . 4 S0CS3 -'- 1 0 7 3 ± 2 . 5 152+1,2 S0CS3 ;- :WT 1 3 0 6 ± 1 . 9 57+1.0 SOCS3 S0CS3-'- SOCS3-A:WT Figure 11. IL-10 inhibition of TNF-a protein expression requires SOCS3 during the early phase of signaling. Macrophage ce l l l ines were der ived from wi ld - type or S O C S 3 " / _ b o n e mar row derived macrophages. The reconstituted cel ls are the S O C S 3 _ / " c lone w h i c h has been transduced w i t h the c D N A for S O C S 3 . C e l l s were st imulated w i t h 100 n g / m L L P S ± 10 n g / m L I L - 1 0 for ( A ) 2 hrs and (B) 4 hrs. T N F - a levels i n the supernatants were measured b y E L I S A . The tables above contain the absolute levels o f T N F - a protein ( p g / m L ) for each set o f data. T h i s data represents 1 o f 4 experiments. 47 3.6 IL-10 inhibits expression of TNF-a mRNA in a SOCS3-dependent manner. Since we observed inh ib i t ion o f T N F - a protein product ion b y I L - 1 0 i n a S 0 C S 3 - dependent manner ( F i g . 11), w e then examined the expression o f T N F - a m R N A i n response to L P S + I L - 1 0 at 2 hours. I L - 1 0 was able to inhibi t T N F - a m R N A expression i n the S O C S + / " macrophages (F ig . 12a, panel A ) , but not i n S O C S 3 7 " macrophages ( F i g . 12b, panel A ) . The same blot was reprobed for G A P D H to conf i rm equal loading ( F i g . 12, panel B ) . Once again, I L - 1 0 d i d not require S O C S 3 protein to inhibi t T N F - a m R N A expression at 4 hours (data not shown). A B TNF-a GAPDH A A r \ LPS LPS+IL-10 0 1 2 1 2 r ^ LPS LPS+IL-10 0 1 2 1 2 hours a. S O C S 3 + / - • H | mm* mm .tarn mm VP VP b- S O C S 3 -'- Figure 12. IL-10 inhibits expression of TNF-a mRNA in a SOCS3-dependent manner. S O C S 3 + / " and SOCS3~'~ macrophages were treated w i t h 100 n g / m L o f L P S + 100 n g / m L I L - 1 0 for 1 and 2 hrs. Samples were col lected i n T r i Z o l and R N A was extracted and resolved b y 1% agarose gel and then subjected to R N A blot analysis w i t h probes for T N F - a . Panel B shows the same blot was reprobed for G A P D H to conf i rm equal R N A i n each sample. Th i s experiment was performed 5 times. 48 3.7 Reconstitution of S O C S 3 cells with W T and mutant S O C S 3 . SOCS proteins have three conserved regions: SOCS-box, SH2 and KIR domains. We retfovirally transduced SOCS3 7" macrophage cell clones with wild type or mutant SOCS3 cDNAs (as described by Yoshimura, A et al in JBC 2000) using infection methods devised for high efficiency gene transfer into macrophage cells [127, 130]. Figure 13A shows a schematic representation of these various SOCS3 domain mutants. The cells were sorted based on GFP expression. Lysates prepared from these cells were examined via western analysis to confirm expression of SOCS3 protein (Fig. 13B, lanes 3-9). Samples then were also prepared from the parental SOCS3 + /" and SOCS3"7" cells (Fig. 13B, lanes 1-2) treated with 100 ng/mL of IL-10 for 1 hour to induce expression of endogenous SOCS3 protein. The expression of each SOCS3 protein was confirmed to be similar to each other and similar to levels of endogenous SOCS3 protein induced by IL-10. 49 Y 2 0 4 Y 2 2 1 a. S0CS3 . WT b. S0CS3 . SH2 c. SOCS3 7": KIR d. S0CS3 . SOCS-box I KIR S H 2 d o m a i n Y 2 0 4 Y 2 2 1 KIR R71E Y 2 0 4 Y 2 2 1 L22D SH2 d o m a i n KIR SH2 d o m a i n Y 2 0 4 F Y 2 2 1 e. SOCS3 7": Y204F KIR SH2 d o m a i n f. SOCS3 7": Y221F Y 2 0 4 Y 2 2 1 F KIR SH2 d o m a i n F i g u r e 13 A . S c h e m a t i c r ep resen ta t ion o f v a r i o u s S O C S 3 p r o t e i n mutan t s . The SOCS3 7": SH2 and SOCS3 7": KIR have a pointtation in the domains, while the SOCS3_ /": SOCS-box has a completely deleted domain. The SOCS3"7": Y204F and SOCS3 7": Y221F have a point mutation in one of the tyrosine residues in the SOCS-box domain. 50 1 2 3 4 5 6 7 8 9 33 kDa (SOCS-3) CO 00 &o GO Figure 13B. Reconstitution of SOCS3 " cells with WT and mutant SOCS3. S 0 C S 3 + / " cel ls (lane 1) and S O C S 3 7 " cel ls (lane 2) were treated w i t h 100 n g / m L L P S + I L - 1 0 for 1 hour to induce expression o f endogenous S O C S 3 protein. The reconstituted cells (lane 3-9) are the S O C S 3 ~~ cel ls w h i c h have been transduced w i t h an empty vector, c D N A for S O C S 3 , S H 2 domain mutant ( R 7 1 E ) , K I R doma in mutant ( L 2 2 D ) , S O C S - b o x delet ion ( D C 4 0 ) , Y 2 0 4 F or Y 2 2 1 F , w h i c h const i tut ively express the myc- tagged S O C S 3 protein as described i n the M a t e r i a l and Methods . Lysates were made I X i n S D S - P A G E sample buffer, resolved b y S D S - P A G E and subjected to immunoblo t analysis w i t h antibody to S O C S 3 protein. The same blot was reprobed w i t h anti-erk ant ibody to show equal protein present i n each sample. These results are representative o f 3 independent experiments. 5 1 3.8 IL-10 requires all domains of SOCS3 protein for inhibition of T N F - a protein production. Once it was conf i rmed that a l l the S O C S 3 7 " macrophages reconstituted w i t h various S O C S 3 domain mutants express comparable levels o f S O C S 3 protein, experiments were conducted to determine w h i c h one o f these domains are important i n the inh ib i to ry effects o f I L - 10 o n T N F - a protein product ion. C e l l s were st imulated w i t h 100 n g / m L o f L P S + I L - 1 0 (10 n g / m L ) for 2 hours. The levels o f T N F - a protein i n culture supernatant were determined v i a an E L I S A . F igure 14 shows that I L - 1 0 required a l l domains o f S O C S 3 protein for i nh ib i t i on o f T N F - a protein product ion. 52 100 (3 O O i- o. a ti Z f- x 60 40 20 0 00 U O 00 o O ' oo o o > oo u O 00 CN X oo ^ ' 00 00 O O 00 O 00 ai 5 m oo <_> O oo oo U O oo X o oo U O oo o r*1 OO U O 00 IX, CN 00 O O oo LPS+IL-10 Cells S0CS3 + /- SOCS.V n S0CS3"' lxlO" 6 lxlO" 6 7 Vector 9xl0" 7 0.80 7 WT 0.80 lxlO" 6 7 SH2 9xl0- 5 0.06 7 KIR 4xl0" 6 0.08 7 SOCS- box 6xl0' 5 0.06 7 Y204F 3xl0" 5 0.18 7 Y221F lxlO" 6 0.07 7 Figure 14. IL-10 requires all domains of SOCS3 protein for inhibition of TNF-a protein production. S O C S 3 + ~ , S O C S 3 7 " and the SOCS3" 7 " reconstituted cel ls (as described i n F i g . 13) were treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 2 hours. The supernatants were col lected to be analyzed for T N F - a secretion b y E L I S A . T h i s experiment was carr ied out 7 independent times and p-values were calculated us ing one-way A N O V A analysis to determine the s ignif icance o f the difference between each SOCS3" 7 " reconstituted cel ls relative to S O C S 3 + / " or S O C S 3 7 " cel ls . These p-values are l is ted i n the table, a p-value < 0.01 was considered significant. 53 3.9 Excluding the KIR domain, IL-10 requires all domains of SOCS3 protein for inhibition of TNF-a mRNA expression. In order to determine the domain o f S O C S 3 i n v o l v e d i n I L - 1 0 inh ib i t i on o f T N F - a m R N A , S O C S 3 + / " a n d S O C S 7 " macrophages, as w e l l as a l l the S O C S 3 7 " macrophages reconstituted w i t h various S O C S 3 doma in mutants were treated w i t h 100 n g / m L o f L P S + I L 1 0 (10 n g / m L ) for 2 hours and total R N A was prepared for a Nor the rn analysis. S O C S 3 7 " : W T - cel ls regained their responsiveness to I L - 1 0 and were able to inhibi t T N F - a m R N A expression, w h i l e the S O C S 3 7 " : V e c t o r behaved s imi la r to parental S O C S 3 7 " cel ls (F ig . 15c, d, respect ively) . F igure 15 also shows that T N F - a m R N A expression is inhib i ted i n S O C S 3 7 " : K I R (15g), but not i n S O C S 3 7 " : S O C S - b o x (F ig . 15e), S O C S 3 7 " : S H 2 (F ig . 15f), S O C S 3 7 " : Y 2 0 4 F ( F i g . 15h), and S O C S 3 7 " : Y 2 2 1 F (F ig . 15i) macrophages. R e p r o b i n g the northern blot for G A P D H m R N A conf i rmed equal R N A loading. 54 F i g u r e 15. E x c l u d i n g K I R d o m a i n , I L - 1 0 r equ i r e s a l l d o m a i n s o f SOCS3 p r o t e i n fo r i n h i b i t i o n o f T N F - a m R N A expres s ion . S O C S 3 + / \ S O C S 3 / _ and the S O C S 3 - / - reconstituted cel ls (as described i n F i g . 13) were treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 2 hours. The R N A was col lected i n T r i Z o l and samples were resolved b y 1% agarose gel and subjected to R N A blot analysis w i t h probe for T N F - a . The same blot was re-probed for G A P D H m R N A to conf i rm equal R N A i n each sample. The bands were quantitated us ing a phosphoimager, and the ratios o f T N F - a m R N A / G A P D H m R N A were plot ted as histograms (right-hand panel). T h i s experiment was carried out 4 t imes. 55 3.10 Y204/Y221 of SOCS3 protein is important for inhibition of NO production by IL-10. W e then examined whether inh ib i t ion o f N O product ion b y I L - 1 0 is also S O C S 3 - dependent. Parental S O C S 3 + / " and S O C S 3 / _ macrophages were treated w i t h 100 n g / m L o f L P S + I L - 1 0 (10 n g / m L ) for 24 hours, and the levels o f N O i n the supernatants were determined us ing the Greiss assay. I L - 1 0 was able to inhibi t N O product ion i n S O C S 3 + / " cel ls , but not S O C S 3 7 " cel ls . T o ensure that this effect is due so le ly to the absence o f S O C S 3 protein, S O C S 3 / _ ce l ls reconstituted w i t h S O C S 3 c D N A were tested as w e l l and F igure 16 shows that reconsti tut ion w i t h wi ld - type S O C S 3 restored responsiveness to I L - 1 0 , w h i l e the S O C S 3 7 " cel ls reconstituted w i t h an empty vector behave l i ke parental S O C S 3 7 " cel ls . N e x t , SOCS3" 7 " cel ls reconstituted w i t h var ious S O C S 3 doma in mutants were examined i n order to determine w h i c h o f these regions are important i n I L - 1 0 ' s ab i l i ty to inhib i t N O product ion. O n l y mutat ion o f Y 2 0 4 and Y 2 2 1 i n the S O C S - b o x doma in abrogated the abi l i ty o f I L - 1 0 to inhibi t N O product ion ( F i g . 16). 56 B O 3 - O O a, O x 40- co y> o o CO « 3 > 00 o o co co o co co CO U O CO a* CO O O 00 co O O co a o co x o (1* o co U O CO r 1 LPs+iL-10 CO U O co cells S'OCS3+/- SOCS3-- SOCS3' 2xl0-6 2xl0"6 7 Vector 6xl0 6 0.70 7 WT 0.70 2xl0"5 7 SH2 0.60 5xl0"3 7 KIR 0.20 9xl0"5 7 SOCS- box 0.20 3xl0"4 7 7 Y204F 4x10° 0 90 / Y221F lxlO"4 0.90 Figure 16. Y204 and Y221 of SOCS3 protein is important for inhibition of NO production by IL-10. S O C S 3 + / " , S O C S 3 7 " and the S O C S 3 7 " reconstituted cel ls (as described i n F i g . 13) were treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 24 hrs. The supernatants were col lected to be analyzed for N O product ion v i a nitrite assay. T h i s experiment was carr ied out 7 independent times and p-values were calculated us ing O n e - w a y A N O V A to determine the signif icance o f the difference between S O C S 3 ' 7 " reconstituted cel ls relative to S O C S 3 + / " or SOCS3" 7 " cel ls . These p-values are l isted i n the table, a p-value o f < 0.01 was considered significant. 57 3.11 IL-10 requires Y204 and Y221 of SOCS3 for inhibition of iNOs protein expression. Since w e observed N O inh ib i t ion b y I L - 1 0 i n a SOCS3-dependen t manner ( F i g . 16), w e investigated whether the inh ib i t ion o f N O b y I L - 1 0 is due to downregula t ion o f inducib le n i t r ic ox ide synthase ( i N O S ) , w h i c h catalyzes the product ion o f N O from L-arg in ine . Parental S O C S 3 + / " a n d S O C S 3 / " cel ls , as w e l l as the S O C S 3 7 " reconstituted w i t h var ious S O C S 3 constructs (as ment ioned above) were treated w i t h 100 n g / m L o f L P S + L + 1 0 (10 n g / m L ) for 24 hours and ce l l lysates Were prepared for determination o f i N O S protein b y immunob lo t analysis. F igure 17 shows that I L - 1 0 was able to inhibi t expression o f i N O S prote in i n S O C S 3 + / " b u t not i n S O C S 3 7 " cel ls and this inh ib i t ion required both Y 2 0 4 and Y 2 2 1 i n the S O C S - b o x domain . The blots were re-probed w i t h antibody against E R K 1 / 2 to conf i rm equal amount o f protein was loaded for each sample. 58 SOCS+/- socs-/- Vector Reconstituted WT r r i r i r 0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 iNOS SOCS-box SH2 KIR Y204F Y221F I I I I I I I 1 I I 0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 0 LPS L+10 ^ .„_ *. r Quantitation of iNOS protein expression 100 iNOS erk :HHHHHH I 1 i | • i F i g u r e 17. IL-10 r equ i r e s Y 2 0 4 a n d Y 2 2 1 o f S O C S 3 fo r i n h i b i t i o n o f i N O s p r o t e i n e x p r e s s i o n . S O C S 3 + / ~ , S O C S 3 7 " and the SOCS3" 7 " reconstituted cells (as described i n F i g . 13) were treated w i t h 100 n g / m L L P S and 10 n g / m L I L - 1 0 for 24 hrs. Lysates were prepared i n I X i n S D S - P A G E sample buffer, resolved b y S D S - P A G E and subjected to immunoblo t analysis w i t h ant ibody to i N O S protein. The same blot was reprobed w i t h antibody to erk protein to conf i rm equal protein load ing i n each sample. The bands were quantitated b y densitometry and the ratios for i N O S protein/erk protein were plotted as histograms (right-hand panel). These are results f rom 3 independent experiments. 59 CHAPTER 4: Discussion The innate immune system initiates loca l and sometimes systemic inf lammatory responses that alert the b o d y to the presence o f potential threats and guides the development o f subsequent adaptive i m m u n e responses. Hence there are two faces to the inf lammatory process; w h i l e o n one hand in f lammat ion is usual ly helpful and protective to the host, i f left unchecked, the excessive amount o f inf lammatory cytokines cou ld cause tissue destruction, phys io log i ca l changes and compl ica t ions such as septic shock. It is s t i l l poo r ly understood h o w an in i t i a l , benef ic ia l host response to infect ion mediated b y inf lammatory mediators, can sometimes progress to a tox ic systemic reaction. L P S is a potent activator o f macrophages and this causes release o f various pro- inf lammatory mediators such as T N F - a , N O , I L - 1 , I L - 6 and I L - 8 , a l l o f w h i c h are suppressed b y I L - 1 0 [100]. Howeve r , the mechan i sm b y w h i c h I L - 1 0 inhibi ts L P S s igna l l ing pa thway is not defined as o f yet. I L - 1 0 induces expression o f m a n y proteins i n a Stat3-dependent manner. One candidate protein whose regulat ion is b y I L - 1 0 i n a Stat3- dependent manner is S O C S 3 . S O C S fami ly o f proteins are t ranscr ipt ional ly activated b y a broad range o f extracellular l igands and functions i n a c lass ica l feedback loop to regulate s ignal transduction through mul t ip le cytokine and growth factor receptors [157, 158, 171]. These proteins were i n i t i a l l y c loned as cy tokine- inducib le immediate-ear ly genes that c o u l d inhib i t act ivat ion o f Stat proteins and b io log i ca l responses to several cytokines [40, 147, 151]. W e and others have found that I L - 1 0 rap id ly induces S O C S 3 m R N A i n macrophages (F ig . 7a) [105] and i n neutrophils [172]. Th i s induct ion occurs i n a Stat3 (F ig . 67b, 7c) and Y 4 4 6 / Y 4 9 6 ( h u m a n - I L - l O R a ) dependent manner ( F i g . 7d, 7e). S O C S 3 protein is induced b y I L - 1 0 alone as early as 1 hour (F ig . 8b). H o w e v e r , stronger S O C S 3 protein expression is observed i n macrophages treated w i t h L P S + I L - 1 0 ( F i g . 8c) and the same is true for S O C S 3 60 m R N A (data not shown). Th i s enhancement m a y be due to post-transcriptional s tabi l izat ion o f the m R N A or post-trahslational regulat ion o f S O C S 3 protein [173]. Other investigators have reported L P S induc t ion o f S O C S 3 protein, however this induc t ion was observed o n l y after 3 hours post s t imulat ion [173], at w h i c h t ime autocrine cytokines l i ke I L - 1 0 are be ing produced. In our hands however , L P S treatment d i d not result i n induc t ion o f S O C S 3 protein at any o f the t ime points examined. S ince mere expression o f S O C S 3 protein i n macrophages was not sufficient to inhib i t T N F - a protein product ion i n response to L P S , this suggests that addi t ional I L - 1 0 signals such as tyrosine phosphoryla t ion , protein modi f i ca t ion or act ivat ion o f other proteins m a y be required. S O C S 3 has been reported to be phosphorylated b y activated J A K s , Src f a m i l y kinases, and receptor tyrosine kinases (eg. E P O R , P D G F R ) at Y 2 0 4 and Y 2 2 1 i n the conserved S O C S - b o x motif . S O C S 3 can b i n d R a s G A P , an inhib i tor o f Ras , w h e n phosphoryla ted b y P D G F , resul t ing i n E R K act ivat ion important for c e l l su rv iva l despite its i nh ib i t i on o f Stat5 phosphoryla t ion due to inh ib i t ion o f I L - 2 s igna l l ing pa thway [168]. Tyros ine phosphory la t ion o f S O C S 3 can ensure c e l l su rv iva l and c e l l cyc l e progression (proliferation) through the Ras pa thway i n this c e l l system. I L - 1 0 induced phosphoryla t ion o f S O C S 3 protein at Y 2 0 4 w i t h i n 2 hours post s t imula t ion o f J774.1 macrophages (F ig . 10a and 10b, respect ively) . The speci f ic i ty o f the phospho-specif ic antibodies raised against Y 2 0 4 / Y 2 2 1 residues were tested o n S O C S 3 7 " : Y 2 0 4 F and S O C S 3 A : Y 2 2 1 F macrophages. Unfortunately, w e were o n l y able to con f i rm the speci f ic i ty o f the phospho-specif ic 204 ant ibody (F ig . lOe). The phospho-specif ic 221 ant ibody also reacted w i t h S O C S 3 7 " : Y 2 2 1 F cel ls ( lOf) , so it either cross-reacts w i t h the S O C S 3 protein or S O C S 3 P Y 2 0 4 . B y reconsti tuting S O C S 3 7 " macrophages w i t h the S O C S 3 Y 2 0 4 F / Y 2 2 1 F double mutant, 61 w e w i l l be able to exclude the latter poss ib i l i ty . Therefore, b y immunob lo t analysis w e cannot conclude whether I L - 1 0 induces phosphoryla t ion at tyrosine 221 o f S O C S 3 protein. H o w e v e r , our analysis w i t h the S O C S 3 - / " : Y 2 2 1 F macrophages suggest that this residue is indeed important for I L - 1 0 inh ib i t i on o f T N F - a and N O product ion. Recen t ly it has been shown that S O C S 3 phosphoryla t ion at Y 2 2 1 , a l lows S O C S 3 to b i n d N e k and C r k - L , adaptor proteins, and recruit N e k to activated receptor tyrosine kinases and modulates N e k tyrosine phosphoryla t ion i n fibroblasts, thus regulat ing adaptor protein s ignal transduction [174]. N e k S H 3 domains are i n v o l v e d i n act ivat ing the I N K and p38 M A P kinase cascades [175], and C r k - L S H 3 domains are i n v o l v e d i n act ivat ing E R K and I N K pathway [176]. Therefore, phosphoryla t ion o f S O C S 3 causes modula t ion i n the regulat ion o f proteins that interact w i t h S O C S 3 protein, and thus affecting the downstream events. Hence it is possible that I L - 1 0 induces phosphoryla t ion o f S O C S 3 protein, w h i c h a l lows S O C S 3 protein to interact and inhib i t proteins that effect transcription and translation o f in f lammatory mediators, such as T N F - a and N O . Inhib i t ion o f T N F - a product ion b y I L - 1 0 has been attributed to effects o n N F K B act ivat ion [88], M A P K s igna l l ing pa thway [94], rate o f t ranscript ion [177], m R N A stabil i ty [142], and translational eff ic iency [94]. W e n o w show that TNF-O! protein product ion and m R N A expression was inhib i ted b y I L - 1 0 i n a SOCS3-dependen t manner, but o n l y up to 2 hours post s t imulat ion (F ig . 11, panel A and F i g . 12, respectively) . W e d i d not see any requirement for S O C S 3 protein at 4 hours post s t imulat ion (F ig . 11, panel B ) , so w e conclude that pr ior to 2 hours, I L - 1 0 inhibi ts L P S s igna l l ing i n SOCS3-dependen t manner, but after 2 hours there m a y be other Stat3-dependent or independent pathways that come into play. 62 4.1. SH2 domain of SOCS3 protein is important in inhibition of TNF-a protein production and mRNA expression by IL-10. I L - 1 0 requires the S H 2 doma in o f S O C S 3 protein for i nh ib i t i on o f L P S - i n d u c e d T N F - a protein product ion ( F i g . 14) and m R N A expression ( F i g . 15f). Poss ib le mechanisms b y w h i c h this m a y happen is discussed be low. A l t h o u g h S O C S 1 binds d i rec t ly to the act ivat ion loop o f Jaks through its S H 2 domain , the S H 2 domain o f S O C S 3 binds to cytokine receptor. S H 2 doma in o f C I S stably associates w i t h the tyrosine-phosphorylated cy top lasmic part o f E P O and I L - 3 receptors [178]. Y 4 0 1 o f E P O receptor is an essential tyrosine site for both S O C S 3 - S H 2 doma in b i n d i n g and Stat5 act ivat ion, suggesting that S O C S 3 inhibi ts ac t iv i ty o f Stat5 through its S H 2 doma in [146]. M u t a t i o n i n the S H 2 domain enhances the LIF-dependent Stat3 and E P O - dependent Stat5 transcriptional act ivi ty [165]. The S H 2 doma in o f S O C S 3 has been shown to b i n d to Y 7 5 7 / 7 5 9 o f g p l 3 0 , Y 9 8 5 o f the lept in receptor, and Y 4 0 1 o f the E P O receptor, some o f w h i c h are the same b i n d i n g sites for SH2-con ta in ing tyrosine phosphatase 2 ( S H P 2 ) [83, 158, 159, 179]. S ince S O C S 3 inhibi ts g p l 3 0 s igna l l ing and S H P 2 promotes g p l 3 0 s igna l l ing through act ivat ion o f mitogen-act ivated protein kinases, thus it is possible that S O C S 3 suppresses aspects o f g p l 3 0 s igna l l ing b y compet ing w i t h S H P 2 for receptor b ind ing . Hence , i n S O C S 3 7 " macrophages reconstituted w i t h S H 2 domain mutant, it is l i k e l y that I L - 1 0 is unable to inhib i t T N F - a protein (F ig . 14) and m R N A expression (F ig . 15f), because loss o f S H 2 doma in o f S O C S 3 m a y a l l o w for S H 2 domain o f S H P 2 or some other SH2-con ta in ing protein to b i n d L P S s igna l l ing targets such as J N K , and promote transcription o f T N F - a and subsequent T N F - a protein expression b y targeting other M A P K s . 63 4.2. KIR domain of S O C S 3 protein is important in inhibition of T N F - a protein production by IL-10. The K I R doma in o f S O C S 3 protein is important for i nh ib i t i on o f L P S - i n d u c e d T N F - a protein product ion b y I L - 1 0 (F ig . 14). In other systems, S O C S 3 has been shown to b i n d to the tyrosine-phosphorylated peptide Y 7 5 9 o f I L - 6 receptor ( g p l 3 0 ) , through its S H 2 domain , w h i c h brings its K I R doma in i n p r o x i m i t y to Jak2 and causes its inh ib i t ion . S O C S 3 has been shown to spec i f ica l ly b i n d to tyrosine 1007 i n the act ivat ion loop o f Jak2, the phosphoryla t ion o f w h i c h is required for its act ivat ion. Thus , S O C S 3 might inhibi t Jak kinase ac t iv i ty through the pseudosubstrate, K I R , b y recrui t ing and b i n d i n g to a c r i t i ca l phospho-tyrosine at the intracel lular part o f a cy tokine receptor. M u t a t i o n i n the K I R doma in enhances the L I F - dependent Stat3 and EPO-dependent Stat5 transcriptional ac t iv i ty [165]. A point mutat ion i n the K I R doma in overcomes the inh ib i tory effect o f bo th S O C S 3 and J A B prote in and transgenic m i c e expressing S O C S 3 mutated i n K I R doma in show more potent Stat3 act ivat ion and a more severe col i t i s induced w i t h dextran sulphate sod ium ( D S S ) [180]. It is possible that i n S O C S 3 7 " : W T macrophages, K I R d o m a i n o f S O C S 3 is able to induce a translational suppression o f T N F - a prote in b y inh ib i t ing an upstream kinase act ivi ty, but m a y be K I R domain ' s role is not extended to transcriptional regulat ion, w h i c h w o u l d exp la in w h y i n S O C S 7 " : K I R , i nh ib i t i on o f T N F - a protein p roduc t ion (F ig . 14), but and not T N F - a m R N A expression ( F i g . 15g) b y I L - 1 0 is disabled. Thus one potential target is the p38 M A P K pathway w h i c h has been shown to be responsible for translational control o f T N F - a prote in [93]. 64 4.3. SOCS-box domain is important in inhibition of TNF-a protein production and mRNA expression by IL-10. I L - 1 0 requires S O C S - b o x doma in o f S O C S 3 protein for i nh ib i t i on o f L P S - i n d u c e d T N F - a protein product ion ( F i g . 14) and m R N A expression (F ig . 15e). In addi t ion to interference w i t h kinase ac t iv i ty and b ind ing to other s igna l l ing molecules , S O C S can also target associated proteins through interaction w i t h the S O C S - b o x for proteasome-mediated degradation. The S O C S - b o x doma in has no catalytic act ivi ty and mediates phys io log i ca l effects o f S O C S 3 through protein-protein interactions [181]. A mode l is proposed where the target molecules conta ining phospho-tyrosine might become a substrate o f the proteolyt ic mach inery b y b i n d i n g to S O C S : after b i n d i n g o f S O C S , the S O C S - b o x acts as an adaptor molecu le , b r ing ing into its complex E l o n g i n B C [160, 163]. The target protein is then ubiqui t inated through recruitment o f the E 3 ligase. D u r i n g the subsequent proteolyt ic association, the substrate and the associated S O C S proteins m a y be destroyed, and the c e l l is ready to respond once again i f the s t imul i are s t i l l present [163]. Ev idence exists that Jaks can be immunoprecip i ta ted i n a complex conta ining S O C S 1 , E l o n g i n B and E l o n g i n C [162]. It has been shown that IL -6 - induced expression o f S O C S 3 is sustained i n the presence o f the proteasome inhibi tor , L L n L , i m p l y i n g that S O C S 3 protein m a y be rap id ly targeted for proteasomal degradation soon after its induc t ion [163]. In support o f these results, us ing ant i -ubiqui t in antibodies, it has been shown that a post- translational mod i f i ed fo rm o f C I S protein ( -37 k D a ) exists i n addi t ion to the predicted i n vi t ro translated protein size o f C I S ( -33 k D a ) . Th i s 37 k D a form accumulates i n the presence o f the proteasome inhibi tors , L L n L and lactacystin, but r ap id ly degrades w h e n the protein synthesis is b l o c k e d b y cyc lohexamide [162, 178, 182]. S O C S protein expression can also inhib i t E P O 65 receptor and Stat5 phosphoryla t ion, w h i c h is not seen i n the presence o f the proteasome inhibi tors ind ica t ing proteasome involvement i n inact ivat ion o f both EPO-recep to r and Stat5 [182]. S ince S O C S - b o x has been shown to be i n v o l v e d i n both s tab i l iz ing and degrading its interacting molecules , it is possible that it m a y serve to s tabi l ize the IKB protein, thus inh ib i t ing N F K B pathway. In alveolar macrophages, I L - 1 0 stabil izes IKB protein b y de lay ing its L P S - mediated degradation and result ing i n delayed nuclear translocation o f the p65 subunit [139]. It is also possible that S O C S - b o x m a y interact w i t h other L P S s igna l l ing proteins such as p38 M A P K , I N K or I K K and cause their degradation i n response to I L - 1 0 , w h i c h w o u l d also result i n i nh ib i t i on o f N F K B pa thway ( invo lved i n transcript ion o f T N F - a and i N O S ) . T h i s m a y exp la in the loss o f inh ib i t ion at T N F - a : protein product ion (F ig . 14) and T N F - a m R N A expression (F ig . 15e) i n macrophages expressing S O C S - b o x mutant. B u t w e do not observe inh ib i t ion o f i N O S protein expression b y I L - 1 0 i n macrophages expressing S O C S - b o x mutant, poss ib ly due to different mechanisms c o m i n g into p l ay at different t imes. Macrophages were st imulated w i t h L P S + I L - 1 0 for 2 hours for T N F - a experiments, w h i l e cel ls were st imulated for 24 hours for the i N O S protein immunoblo t . F o r inh ib i t ion o f T N F - a prote in i n response to L P S , the role o f S O C S - b o x m a y be cr i t ica l for suppression o f the targets o f I L - 1 0 , but for i nh ib i t i on o f i N O S protein expression, S O C S - b o x doma in m a y o n l y p l ay a secondary role, w h i c h can be compensated for b y the other domains w h e n S O C S - b o x d o m a i n is inaccessible to I L - 1 0 . 4.4. Y204 and Y221 of SOCS3 are important in inhibition of NO production and iNOS protein expression by IL-10. N O mediates the ab i l i ty o f macrophages to k i l l or inhibi t the growth o f tumour cel ls , bacteria, fungi and parasites [45]. B u t this regulat ion must be t ight ly cont ro l led since unregulated N O product ion causes fa l l i n b l o o d pressure (vasodilat ion) , w h i c h potent ia l ly 66 results i n injury to host tissue. Here w e show that I L - 1 0 inhibi ts N O product ion i n S O C S 3 - dependent manner and o n l y requires Y 2 0 4 and Y 2 2 1 o f S O C S 3 protein to be intact ( F i g . 16). W e further examined the effect o f S O C S 3 i n response to I L - 1 0 o n i N O S , the enzyme that is responsible for product ion o f N O . I L - 1 0 inh ib i t ion o f i N O S prote in expression reflected the results obtained for N O product ion. i N O S prote in expression was inh ib i ted b y I L - 1 0 i n S O C S 3 - dependent manner and o n l y requi r ing the Y 2 0 4 and Y 2 2 1 o f S O C S 3 protein (F ig . 17). Th i s observat ion is supported b y another study carried out b y O ' F a r r e l l et a l , where they have demonstrated that I L - 1 0 inhibi ts N O product ion and i N O S prote in expression i n a Stat3- dependent manner [127]. Interestingly I L - 1 0 is able to inhib i t N O product ion and i N O S protein expression w h e n the entire S O C S - b o x doma in is deleted ( S O C S 3 7 " : S O C S - b o x ) , but not w h e n there is a point mutat ion i n either o f the two tyrosine residues, 204 ( S O C S 3 7 " : Y 2 0 4 F ) and 221 (SOCS3" 7 " : Y 2 2 1 F ) , located i n the S O C S - b o x domain (F ig . 16 and F i g . 17). T h i s interesting observat ion c o u l d be expla ined b y the f o l l o w i n g mode l : w h e n the S O C S - b o x doma in is deleted, S O C S 3 is not able to interact w i t h E l o n g i n B / C complex and target proteins for ubiqui t ina t ion , but the other domains o f S O C S 3 ( S H 2 / K I R domains) are s t i l l capable o f ca r ry ing out inh ib i to ry effects o n protein X ( in the L P S s ignal ing pathway). F o r instance, S O C S 3 protein can inhib i t the associat ion o f protein X w i t h other molecules b y interacting w i t h the phospho-tyrosines o f prote in X through its S H 2 domain , or i f protein X is a kinase, then S O C S 3 can suppress its kinase act ivi ty v i a the K I R domain . Howeve r , i n S O C S 3 / " : Y 2 0 4 F and S O C S 3 7 " : Y 2 2 1 F macrophages, S O C S - b o x domain is intact and can s t i l l associate w i t h E l o n g i n B / C complex , but S O C S 3 cannot be phosphorylated b y I L - 1 0 since there is point muta t ion i n either Y 2 0 4 or Y221. Thus w h e n S O C S 3 / E l o n g i n B C complex interacts w i t h a target prote in X, it m a y 67 enhance the stability of protein X rather than degrading it becauseY204/Y221 cannot be phosphorylated by IL-10 and thus cannot be targeted for ubiquitination. Thus, when SOCS3 protein interacts with Elongin B/C and there is no "degradation signal" (ie. phosphorylation by IL-10), then this interaction serves to stabilize the SOCS3 protein and its associated protein. As well, this makes the other domains of SOCS3 protein unavailable and inaccessible for other inhibitory mechanisms, since they are in complex with Elongin B/C. Therefore we observe complete loss of inhibitory effects of IL-10 on NO production and iNOS protein expression in SOCS3"7": Y204F and SOCS3"A: Y221F SOCS3, but not when SOCS-box domain is deleted. 68 4.5. Summary In this study we have successfully demonstrated the importance of SOCS3 protein in mediating the anti-inflammatory action of IL-10, which is also supported by the SOCS3 over- expression study [3]. Our results also show that inhibition of TNF-a and NO production by IL- 10 is mediated by different domains of SOCS3 protein, which suggests that there are different mechanisms that come into play in response to IL-10 (Fig. 18). Table 1 summarized the requirement for different SOCS3 domains in mediating IL-10 inhibition of TNF-a protein production and mRNA expression, as well as NO production and iNOS protein expression. Table 1. Summary of the requirement for different SOCS3 domains in mediating IL-10 inhibition of various macrophage responses. SOCS3 domain TNF-a protein production TNF-a mRNA NO production iNOS protein expression KIR Yes No No No SH2 Yes Yes No No SOCS-box Yes Yes No No Y204 Yes Yes Yes Yes Y221 Yes Yes Yes Yes 69 Figure 18. Schematic representation of the mechanism by which IL-10 may be inhibiting L P S signaling pathway. IL-10 induces S O C S 3 protein expression, wh ich then inhibits L P S signaling at multiple levels by different domains. IL-10 requires S H 2 domain, S O C S - b o x domain, Y 2 0 4 and Y221 o f S O C S 3 protein to inhibit T N F - a m R N A expression possibly by interfering wi th N F K B protein activation. S O C S 3 requires a l l its domains for inhibit ion o f T N F - a protein release by IL-10 , possibly by interfering either wi th the M A P K s , or IKB protein stability or receptor tyrosine activity. IL-10 requires Y 2 0 4 and Y 2 2 1 o f S O C S 3 protein f o r inhibit ion o f i N O S protein expression by IL-10, wh ich is possibly either through inhibit ion o f I L K pathway or N F K B pathway, and ultimately inhibi t ion o f i N O S protein expression leads to inhibit ion o f N O production. 70 4.6 O t h e r S tud ies O u r hypothesis that S O C S 3 is i n v o l v e d i n the ant i - inf lammatory act ion o f I L - 1 0 disagrees w i t h conclus ions reached b y L e e and C h a u [131]. T h e y observed no effect o f S O C S 3 delet ion (through antisense ol igonucleot ides) on the ab i l i ty o f I L - 1 0 to inhibi t T N F - a product ion. H o w e v e r , their experimental system differs f rom ours i n that they add I L - 1 0 to cel ls 4 hours pr ior to L P S challenge. W e show that I L - 1 0 requires S O C S 3 o n l y i n the early phase (< 2 hours) o f i nh ib i t i on o f T N F - a protein product ion. In their system another IL -10 - induced gene ca l led heme oxygenase-1 ( H O - 1 ) appears to be central to media t ing macrophage deactivation. H O - 1 is not induced unt i l 3 hours after I L - 1 0 s t imulat ion and m a x i m a l levels are not achieved un t i l 24 hours; w h i c h is w h y they chose to add I L - 1 0 four hours p r io r to L P S st imulat ion. W e and others [3, 94] have chosen a system where I L - 1 0 and L P S are added s imul taneously since the phys io log i ca l target o f I L - 1 0 is the activated macrophage. The rest ing macrophage is different f rom the L P S activated macrophage, and it might be reasonable to expect different mechanisms to come into p lay under different circumstances. In contrast, studies carried out b y Jung et a l showed that al though H O - 1 gene is upregulated b y I L - 1 0 ( in D N A array), a specif ic H O - 1 inhib i tor z inc-pro toporphyr in ( Z n P P ) was not able to abrogate I L - 1 0 inh ib i t ion o f L P S - induced T N F - a protein product ion [183]. T h i s suggests that other I L - 1 0 regulated genes are media t ing the ant i - inf lammatory effects o f I L - 1 0 . O u r demonstrat ion o f a role o f S O C S 3 i n I L - 1 0 inh ib i t i on o f macrophage funct ion is also i n apparent contrast to the results obtained b y H . Y a s u k a w a et a l , where they have shown that w h e n S O C S 3 gene is disrupted i n macrophages, there is no affect o n I L - 1 0 ' s ab i l i ty to inhib i t T N F - a as compared to S O C S 3 wi ld - type macrophages [173]. Once again, their system 71 is very different, as they treated their cells w i t h L P S + I L - 1 0 for durat ion o f 5 to 25 hours and then examined for affect on T N F - a product ion. A s w e have shown, I L - 1 0 does not require S O C S 3 to inhibi t T N F - a product ion dur ing the late phase o f s igna l ing (> 2 hours) ( F i g . 11, panel B ) and m R N A expression (data not shown). Thus S O C S 3 protein is important dur ing the early inh ib i to ry act ion o f I L - 1 0 on T N F - a expression b y a macrophage, w h i l e other regulatory mechanisms come into p l ay at later t imes. H o w e v e r , i n support o f a role for S O C S 3 i n I L - 1 0 inh ib i t i on o f in f lammat ion S u z u k i et al have demonstrated that interference w i t h S O C S 3 function i n a transgenic mouse enhanced the mouse ' s suscept ibi l i ty to intestinal in f lammat ion suggesting that the ablat ion o f S O C S 3 does indeed interfere w i t h the no rma l negative regulatory act ion o f I L - 1 0 i n control o f co lon in f lammat ion [180]. 72 CHAPTER 5 : Future Directions One o f the prior i t ies i n further understanding the mechan i sm b y w h i c h S O C S 3 mediates the ant i - inf lammatory act ion o f I L - 1 0 is to identify the proteins w h i c h interact w i t h the var ious domains o f S O C S 3 . The effect o f S O C S 3 cou ld be examined i n response to I L - 1 0 o n ac t iv i ty o f L P S s igna l l ing molecules such as I L K , I K K , I K B , and P K B . Studies w i l l be designed w h i c h w i l l make use o f glutathione-S-transferase (gst) fusion proteins conta ining the different regions o f S O C S 3 (for example, S O C S - b o x domain , S H 2 doma in and the N - t e r m i n a l doma in w h i c h includes the 22 amino ac id K I R domain . In case o f the K I R domain , the candidate protein cou ld be a kinase, such as the src tyrosine kinase fami ly member L y n , w h i c h is activated b y L P S and inhib i ted b y I L - 1 0 i n monocytes [184]. A s w e l l , the tyrosine phosphoryla t ion sites 204 and 221 located i n the S O C S - b o x doma in are found to be important i n supporting I L - 1 0 inh ib i t ion o f T N F - a product ion and m R N A expression, as w e l l as N O product ion and i N O S protein expression. Exper iments w i l l be designed to identify the proteins that associate w i t h these phosphotyrosyl sites us ing biot inyla ted phosphopeptide corresponding to the relevant tyrosine phosphory la t ion site as the affinity reagent. The biot inyla ted phosphopeptide w i l l be pu l l ed d o w n us ing streptavidin agarose and the associated proteins w i l l be v i sua l i zed b y s i lver staining. I f the bands cannot be ident i f ied through a k n o w n antibody, then proteins w i l l be pur i f ied for microsequence analysis. Once identif ied, the interaction o f these k n o w n or nove l proteins w i t h intact S O C S 3 prote in can be conf i rmed b y co- immunoprec ip i ta t ion studies and their role i n I L - 1 0 s igna l l ing tested b y R N A i - m e d i a t e d k n o c k - d o w n or over-expression studies. F i n a l l y us ing micro-ar ray analysis, w e 73 c o u l d identify other L P S - i n d u c e d genes that are inh ib i ted b y I L - 1 0 i n a SOCS3-dependen t manner. 74 C o n c l u d i n g r e m a r k s W e have def in i t ive ly shown the importance o f S O C S 3 protein i n the ant i - inf lammatory act ion o f I L - 1 0 and also have shown that inh ib i t ion o f N O and T N F - a b y I L - 1 0 is mediated b y different domains o f S O C S 3 protein, w h i c h suggests that there are different mechanisms that come into p lay i n response to I L - 1 0 . Th i s is the first demonstrat ion o f alternate mechanisms o f act ion o f S O C S 3 prote in on divergent pathways activated b y the same s t imul i . A better Understanding o f these s igna l l ing pathways w i l l a l l o w us to d iscover therapeutic targets i n hopes o f a cure for m a n y o f the inf lammatory diseases that affect m i l l i o n s o f ind iv idua ls i n our society. 75 B i b l i o g r a p h y 1. 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