Production, regulation and role of nitric oxide in glial cells.

The central ne rvous system (CNS) consists of tw o major ce ll type s, neurones and glial ce lls. Neuronal communication w ith other neurone s or glial c ells is effected mainly through ne uro-transmitte rs and peptides, w hile glial ce lls appear to use an abundant range of factors for the communication w ith either ne urone s or other glial c ells. The communication betw een ne urone s can span large distances in the body, w hile glial c ell communication is mainly local or paracrine . During the past decade it has become clear that the glial c ells named afte r glue (= glia) have more functions besides acting as a “nerve-glue” to form the brain. ,2 The glial ce lls appeared to be e ssential for ne uronal prote ction, survival and outgrow th during deve lopment and for the neuronal degeneration and regeneration under pathological conditions. In this review we summarize glial ce ll functions and focus on the role , production and regulation of nitric ox ide (NO), a molecule w hich has be en show n to be involved in various ne uroimmune proce sse s.


Introduction
The ce ntral ne rvous syste m (CNS) c onsists of tw o major ce ll type s, ne urone s and glial ce lls. Neuronal communic ation w ith other ne urone s or glial c ells is effecte d mainly through ne uro-transmitte rs and peptides, w hile glial ce lls ap pear to use an abundant range of factors for the communication w ith either ne urone s or other glial c ells. The communic ation be tw e en ne urone s can span large distanc es in the body, w hile glial c ell c ommunic ation is mainly loc al or paracrine . During the past decade it has be c ome clear that the glial c ells name d afte r glue (= glia) have more functions be sides acting as a "ne rve-glue" to form the brain. 1 ,2 The glial ce lls appe are d to be e sse ntial for ne uronal prote ction, survival and outgrow th during development and for the ne uronal dege ne ration and re ge ne ration under pathological c onditions. In this re vie w w e summarize glial ce ll functions and foc us on the role , produc tion and re gulation of nitric ox ide (NO), a molec ule w hich has be en show n to be involved in various ne uroimmune proce sse s.

Glial Cells
Glial cells can be divided into microglial ce lls and macroglial ce lls, the latte r of w hich are subdivided in astroglial c ells and oligodendrocyte s. The oligodendroc ytes are know n for the ir myelin production that w raps the ax ons in the w hite matter of the brain, and are affec te d in disease s like multiple scle rosis (MS). Glial ce ll activation has be en indic ated in many ne uropathologic al disease s like Alzheimer's disease , 3 -7 Parkinson's disease, 5 multiple scle rosis , 8 acquire d immune de ficie ncy syndrome (AIDS) deme ntia c omplex (ADC) 9 -11 and other disorde rs.

Microglial cells
Mic roglial c ells, formerly name d Hortegaglia or Mesoglia, have be en de scribe d in de tail for the first time in 1932 by De l Rio-Horte ga 1 2 cons idere d as the fathe r of mic roglia. Mic roglial ce lls are ubiquitously distribute d in the CNS, show he te roge ne ous morp hology and comprise up to 20% of the total glial c ell population in the brain. 13 It w as Del Rio-Hortega w ho propose d that mic roglial cells oc cur in tw o morphologically distinc t forms, the ameboid or macrophage -like form re prese nting as ac tive microglial c ells seen in de ve loping brain and at site s of injury. The se ce lls convert into the highly branched ramifie d microglial ce lls 14 -1 7 vie w e d as quiesce nt ce lls in the mature CNS 1 8 w hich e ventually can transform into ac tive mac rophages (reac tive mic roglial c ells) (Fig. 1). 19 ,20 De l Rio-Horte ga suggeste d that microglial c ells served as macrophage s or phagoc ytic ce lls, w hich has found substantia l support. 21 -2 3 The name mesoglia w as de rive d from the ir proposed mesodermal origin. Indee d more re c ent and elaborative studie s support a bone-marrow origin 17 ,2 4 from the usual macrophage pre cursor. Blood monocytes or monoc yte pre cursors invade the brain during development. 25 ,26 In studie s using chimeric rats, support w as found for the bone marrow origin hypothe sis 27 ,2 8 but in othe r rat chimera ex periments the contrary w as found. 29 ,30 The pre vailing c onc ept is how e ver that blood monoc ytes are the pre c ursors of ameboid mic roglial c ells. The re is appare ntly no ne e d for the influx of a large numbe r of monocyte s into the brain pare nchyma unde r normal conditions, sinc e mic roglial c ells have an ex treme long life and have a low turnove r rate . 31 ,32 The ir numbe rs can be augme nted by local prolife ration 1 9,3 3 -36 or by immigration of blood monoc ytes. 37 Unde r pathological c onditions inc re as ed monocyte infiltration into the brain parenchyma is found 8 ,38 ,39 and the influx is sugge ste d to be mediated by the mic roglial c ell derived chemokine MCP-1 4 0 or adhe sion mole cules.

Mic ro g lia l ce ll fu n ctio n s
The mic roglial ce lls serve as immunore gulatory cells, and are essential for re sistance to inter-and intrace llular pathogens . The mic roglial cells are p re sent in a re sting or a ramifie d form (Fig. 2) and are very important in immunosurve illance . Afte r activation of the re sting ce lls the microglial ce lls ex hibit a highly pote nt phagocytic ac tivity of fore ign organisms and material, phagocytosis of injured or ne crotic tissue, antimic robial immunity, elimination of tumour cells and re gulate inf lammatory re sponse s. 41 Mic roglial ce lls have be en re porte d to posse ss Fc re ce ptors, 1 8,4 2 CD4 antige n 43 and major histocompatibility complex (MHC) class I and II antigens 41,44 -46 and thus are antigen pre se ntating ce lls. Inte rferon-g and inte rleukin-4 (IL-4) up-re gulate MHC class II ex pre ssion and induc e mic roglial ce ll p roliferation . 47 In addition, mic roglial c ells show che motax ic activity, like monocytes and macrophage s, to se veral immunologic al factors such as compleme nt factor C5a and to transforming grow th fac tor b (TGFb ) sugge sting that these ce lls c an move to site s of injury and the re by participate in an inflammatory re sponse . 12 ,4 2 ,4 8, 4 9 These observations have le d to a re definition of the brain as immune privileged site a c oncept based on lack of inflammatory re sponses through the absenc e of T and B c ells.
Microglial ce lls share many functional characte ristic s w ith c ells of the monocytic lineage . Inte rleukin-1 (IL)-1 production by mic roglial c ells has be e n demonstrate d for the first time by Giulian ,50 and later by many othe rs 4 1,5 1-54 and IL-1 is found to be pre sent in injure d brain tissue. 5 0,55 Othe r cytokines produc ed by mic roglial cells are TNF-a , 54 ,56 ,5 7 IL-6 5 7-59 and TGFb 6 0 -6 2 and the y produc e p rostanoids such as p rostaglandin E 2 , (PGE 2 ), PGD 2 , thrombox ane 6 3,6 4 and le ukotriene s, LTB 4 , LTC 4 and 5-HETE. 65 IL-5 w as found to be produce d by microglial c ells in v itro w hich may be involved in the inte raction be tw e en glial c ells and immune c ells in the brain . 66 IL-10 and TGFb both immunosuppre ssive and anti-inflammatory cytokines, have be e n de monstrated to be produc ed by human mic roglial cells and dow n-re gulate mic roglial ce ll func tions. 6 0,67 -7 1 The se propertie s confirm that mic roglial ce lls c an initiat e and re gulate immune and inflammatory re sponses w ithin the brain.

Astroglial cells
Astrocyte s, oligodendrocyte s and ne urons are of ec tode rmal origin, and derive from the ne uroep itelium of the primitiv e ne ural tube. Astroglial c ells, unlike ne urone s, re tain the ability to divide throughout life . 7 2 The multipotential ste m c ell de ve lops into the bipote ntial proge nitor ce ll and finally the glial lineage -re stric te d p rogenitor c ell w hich c an differentiate into the oligodendroc yte, the astrocyte type 1 and type 2. 73 -7 6 The astrocytes outnumbe r ne urones 10:1 in mammalian brain, and as their name imply, the y have a star-shape d morphology. The astrocytes can be identifie d by using spe cific marke rs such as glial fibrillary acidic prote in (GFAP), and glutamine synthase (GS), that are spec ific for both types of astroglial cells. 7 7-81 Each c ell forms p roc esses that contac t the blood vessels, w here the y form the so-c alled end-feet or sucker proce sse s w hich also forms part of the bloodbrain barrie r (BBB) toge the r w ith endothe lial cells and the lamina basalis. 7 2 Astroc ytes in the w hite matte r are re fe rre d to as fibrous astroc ytes, w ith numerous fibrils w ithin their cytoplasm. In the gre y matte r, the astroc ytes ge ne rally c ontain few fibrils and are c alled protoplasmic astrocyte s. Inte re stingly, in v itro also tw o type s of astroc ytes can be identifie d, type 1 and type 2 astroc ytes w hich are thought to be in v itro analogous of the protoplasmic and fibrous astrocyte s re sp ective ly 82 (Fig. 2).
As tro g lia l ce ll fu n ctio n s Initially the func tion of astroglial c ells w as thought to be a structural support w ithin the CNS, w ith their p roc esses having junc tions w ith other astroglial c ells, e ndothelial c ells and ne urone s. In addi-Mediators of Inflammation · Vol 7 · 1998 tion in re pair mechanisms the astroglial cells fill open spac es by prolife ratin g and there by forming a glial scar. 83 -86 It is now know n that astroglial ce lls are very important c ells in the outgrow th and survival of ne urone s during development and in ne uropathology. Astroglial c ells produce ne rve grow th fac tor (NGF) 8 7 in v itro and in v ivo and the production of NGF is inc re as ed by IL-1, 8 8 -9 2 TNFa , 9 3 IL-4 and IL-5. 94 Other ne urotrophic fac tors p roduced by astroglial ce lls are ciliary ne urotrophic factor (CNTF), brain-de rive d ne urotrophic factors (BDNF) and fibroblast grow th factor (FGF). 95 ,96 Astroc ytes p roduce c ytokine s like IL-1, 41 ,9 7 -1 00 IL-6, 10 1 IL-3, 10 2 TGFb 6 0,6 2 ,1 0 3 -10 5 and IL-15 10 6 and factors like prostaglandin E 2 (PGE 2 ), 97 ,9 9 ,1 07 ,1 0 8 granuloc ytemacrophage colony-stimulating factor (GM-CSF) 1 09 ,1 10 and microglial mitoge ns (MM). 11 1 TNFa is also produce d by astroglial c ells in vitro afte r lipop olysaccharide (compone nts of Gram-ne gative bacte rial outer membranes; LPS) 56 ,11 2 and IL-1b stimulation 59 , 10 6 or myc oplasma infection. 1 08 Astroc ytes can be induce d to ex pre ss MHC I and II class molecules in v itro and are able to pre se nt antige n 113 ,1 14 and thus are important immune ce lls in the brain. How ever, in contrast to mic roglial cells the y do not ex pre ss significant le vels of MHC class II molecules 11 5 in v ivo .

Glia-glia interactions
Rec ent studie s have show n various interac tions be tw e en mic roglial c ells and astroglial ce lls. IL-1 produce d by microglial c ells has be e n show n to stimulate astroglial c ell proliferation in v itro . 11 6 -11 9 Intrace re bral inje ctions of IL-1 or local production of IL-1 by mic roglial c ells elicit as trogliosis w hich may re sult in scar formation 1 20 ,12 1 and there by have a ne gative effe ct on ax onal outgrow th and re mye lination. In addition the microglial ce lls influe nce the production of NGF by astroglial c ells, w hich is enhance d after IL-1 and IL-5 both produced by mic roglial ce lls. 66 ,94 ,12 2 On the other hand, astroglial cells influenc e mic roglial ce ll func tions. Interleukin-3 (IL-3) is mitogenic to mic roglial ce lls and has be e n suggeste d to be produc ed by astrocyte s. 1 02 , 12 3 Mitotic activity of mic roglial c ells can additionally be e le vated by c olony stimulating fac tor-1 (CSF-1), w hich production is inc re as ed by IL-1, TNFa , 12 4 granulocyte -mac rop hage colony-stimulating fac tor (GM-CSF) 41 ,1 0 9,1 1 0,1 22 ,1 2 5 - 12 7 or microglial mitogen (MM). 1 11 The mic roglial c ells undergo morphological changes in re sponse to factors re le as ed by or through cellcell c ontac t w ith astrocyte s. The se factors de rive d from as trocyte s, induce microglial c ells to be come the ramified, func tionally re sting ce lls in v itro , w hile inflammatory mediators like inte rferon g (IFNg ) and LPS induce mic roglial c ells to be come ameboid. 1 28 , 12 9 Also, blood monocyte s and sple en mac rophages differe ntiate into ramified microglial c ells w hen c ultured onto an as troglial c ell monolayer. 1 30 , 13 1 This illustrates the capac ity of astroglial ce lls to transform ce lls from monocytic origin into c ells w ith mic roglial ce ll morphology.
In addition, as troglial c ells have be en show n to functionally re gulate mic roglial c ell activity. The endotox in induce d synthe sis of iNOS and re le as e of NO but not the production of IL-1b by microglial ce lls is inhibited in the pre senc e of astroglial cells in v itro . 51 Appare ntly both glial c ells communic ate through the production of various factors including cytokines and grow th fac tors. In this w ay the glial ce lls appear to tightly re gulate each others morphology, ac tivity and se cre tion of p roducts .

Glia-neurone interactions
The pre senc e of glia-derived cytokines in the CNS and the func tion of the se c ytokine s in vitro suggest that the y are important for normal brain development and home ostasis. 13 2,13 3 How e ve r, ex ce ssive ex pre ssion of these c ytokine s may be a fac tor in abnormal glial functions leading to ne uropathological e ve nts.
In gene ral astroc ytes have be en found to ex pre ss ne urotrophic factors such as ciliary ne urotrop hic factor (CNTF), ne urotrophin-3, fibroblast grow th factor (FGF) and NGF ne ar the site of injury. 1 34 ,1 35 In v itro ex perime nts show that astrocytes prote ct dopamine rgic ne urons against H 2 O 2 tox icity 136 through actions of glutatione . Intere stingly, survival of the se dopamine rgic ne urones in vitro is enhanc ed by the pre senc e of glial c ells derive d from striatal astroglia: the targe t-de rive d astroglial cells, illustrating astroglial ce ll he te rogene ity. 13 7,1 38 The survival of dopaminergic ne urone s is promote d by glial ce ll-line d de rive d ne urotrophic factor (GDNF) in v ivo . 1 39 In v itro cocultures of ne urone s and as troc yte s induc ed an inc re as ed ce ll survival and ne urite outgrow th 14 0 -14 2 and ax ons might trigger glial diffe re ntiation. 14 3 The ability of astrocyte s to augme nt ne uronal survival w as inc re as ed after treatme nt of the as trocyte s w ith macrophage condition ed medium 14 4 indicating that factors produc ed by macrophage s induc e the production of ne urotrophic fac tors.
Ne urone s are less sensitive to ox yge n or gluc ose deprivation or treatme nt w ith glutamate w he n co-cultured w ith astrocyte s. 1 45 In addition astrocytes inc re as e ne uronal survival unde r pathologic al c onditions sinc e the y have an ene rgy re se rve store d as glycogen w hich be comes available for ne urones under c onditions of e ne rgy substrate limitations. 14 6 Astroglial ce lls are important in the me tabolism of glutamate and GABA and other ne urotransmitte rs, 14 7 and maintain the microenvironme nt by re gulating the ionic composition of the ex tracellular space around the ne urone s. 14 8 Ne urotrophic e ffec ts i.e. ne uronal survival and ne urite ex te nsion have also be e n re p orte d by mic roglial c ell c onditione d medium 1 49 and more spec ifically by the produc tion of NGF 1 50 and thrombospondin. 15 1,15 2 In addition se cre tion of IL-6, IL-1, FGF, TGFb , TNFa by mic roglial c ells and as troglial cells may stimulate ne rve grow th fac tor production by astroglial cells for the re ge ne ration of ne urone s. 9 3,1 3 4,1 53 -1 58 The se fac tors also dire ctly improve the survival of ne urone s and/or have syne rgistic effects w ith NGF. 1 59 ,16 0 While in gene ral factors re lease d by astroglial c ells actually inc re ase the survival of ne urone s, 16 1 mic roglial ce lls in c ontrast, c an direc tly participate in ne uronal ce ll de ath through the re le as e of ne urotoxins. 1 62 Mic roglial ce lls have be en re p orte d in the pre senc e of de gene rating ne urones in various re gions in the brain. 1 8,1 63 -1 65 In the se re gions the mic roglial ce lls c le arly c ontribute to the re moval of p ycnotic ce ll bodie s. Prior to this sc avenge r role , the active participation of microglial ce lls in ne urite amp utation has be en show n on elec tron mic roscopic p ictures of mic roglial c ells e ngulfing ax on proc esses w hich display no obvious signs of de gene ration. 1 66 It is there fore thought that interac tions be tw een ne urones and microglial ce lls may not be re stricte d to ce ll debris sc avenging but mic roglial ce lls may also induc e ne uronal c ell death.
In vitro studies have demonstrate d the production of many differe nt ne urotox ins by microglial cells. Activated microglial ce lls re lease se ve ral c ytotox ic compounds i.e. re ac tive ox ygen intermediate s, 1 67,16 8 NO, prote ase s 5 1,1 69 -1 72 and inflammatory cytokines i.e. IL-1, TNFg or TNFa , 50 ,5 6 ,1 19,1 2 7,1 57 ,1 73,1 7 4 that play a role in ne uronal damage in the CNS. Finally mic roglial ce lls produc e large amounts of glutamate and aspartate in v itro . 17 5 The re lease of these ex citatory amino acids points to a furthe r role of mic roglial ce lls in NMDA re c eptor-mediate d ne uronal injury. 1 75 In addition, cultured microglial c ell re le ase large amounts of H 2 O 2 , w hich leads to ne uronal c ell death in ne uronemicroglial ce ll c oculture s. 1 72,1 7 6,1 77 Taken togethe r, activate d microglial c ells display a broad re pertoire of cytotox ic func tions w hich c ould be involve d in tissue damage during CNS injury. In addition, microglial cells activate astroglial c ells in a w ay that be ne fits re gene ration. Further, the effe ct of ne urotox ic fac tors re le ase d by microglial ce lls can be atte nuate d by prote ins re le as ed from astroglial ce lls. 17 8,1 79 This illustrate s the fascinating and de lic ate inte rac tions that ex is t be tw een glial c ells and ne urone s, w hich are cruc ial in maintainin g ne ural functioning and inte grity. The se studies have contribute d tow ards a c oncep t w hich considers re active mic roglial cells as an opposing forc e to ne urotrophic astroglia, the tw o glial ce ll populations rivaling in re gulating survival of ne urone s. 18 0

Nitric Oxide in the CNS
Various c ell types in the CNS, i.e . ne urone s, e ndothelial cells, microglial cells and as troglial c ells p roduc e NO. Diffe re nt is oforms of the nitric ox ide synthas e (NOS) are re sponsible for the produc tion of NO by these c ell type s. NO in the brain is multipote nt and is re sponsible for blood flow re gulation, may ac t as a ne urotransmitter or as a ne urotox ic agent, de pending on the cellular sourc e, amount and production site .

Isoforms of nitric oxide synthase
NO, a fre e radical gas, w as found to be re sp onsible for the vasodilatation in arterie s and at first name d endothelium derived re lax ing fac tor (EDRF). 1 81 Late r the sourc e of NO w as e luc idate d re vealing the enzymatically c onvers ion of L-arginine to L-citrulline by NO synthase w here by NO is produced (Fig. 3). 18 2 Thre e isoforms of NOS, enc ode d by diffe re nt gene s 1 83 have be e n characte rized, isolate d and cloned to furthe r study the physiologic and/or pathologic functions of NO. 18 4 All thre e isoforms, endothelial (eNOS, ec NOS or type III), constitutiv e (cNOS, nNOS, bNOS or type 1) found in astroglial ce lls and ne urone s and induc ible (iNOS, mNOS, macNOS or type II), NOS are found in the CNS and play a role in ce rtain physiological or pathologic al functions of the CNS (se e follow ing sec tion). 18 5 Constitutiv e NOS is c onstitutiv ely ex pre sse d in ne urone s, is Ca 2+ and c almodulin depe nde nt, and me diates the p roduction of only small amounts of NO afte r stimulation. This ne uron de rive d NO is ve ry rapidly produced and re le as ed since cNOS is constitutiv ely ex pre sse d and doe s not re quire mRNA synthesis, acts as a ne urotransmitte r w ith propertie s that diffe r from othe r ne urotransmitte rs: (a) it is not stored in ve sicles, (b) there are no specific re lease or uptake mechanisms and (c ) its transmission is not synaptic . NO diffuse s into the targe t ce ll and dire ctly re gulates e nzyme s syste ms, such as activation of guanylate c yclase , re sulting in incre ase d cGMP le vels. 1 86 ,1 87 NO has a half life of a few sec onds in contrast to the millise conds of the ne urotransmitters in classical synapses. 18 8,1 89 It is important in ne urotransmission, and NO is c onside re d a candidate for a memory-re lated p roc ess name d long-te rm-pote ntiation (LTP). 19 0,1 91 Indee d, inhibitors of NOS can block LTP. 19 2,1 93 In areas such as the c ere bral cortex , hipp oc ampus, ce re be llum and c orpus striatum, cNOS ex pre ssing ne urone s c ompose 1-2% of all ne uronal ce lls. 19 4 Ac tivation of cNOS in astroglial ce lls w as obse rved after challe nge w ith calcium ionophore s, bradykinin or glutamate. 18 5 Endothe lial NOS (eNOS) produce d by e ndothelial ce lls is a c onstitutiv e Ca 2 + -depe ndent enzyme that is essential for the control of vascular tone . NO transduc es a signal from the endothe lial c ell to the vascular smooth muscle e ve ntually le ading to cGMP production and vasodilation. In the brain, e NOS-de rive d NO re gulates ce re brovasc ular blood flow. 1 95 Induc ible NOS (iNOS) is a Ca 2 + and c almodulin indepe nde nt enzyme . It re quire s ge ne transcription, is slow ly produced and is ac tivated only under pathologic al situations w here mic roglial c ells and macrophage s ex e rt cytotox ic effe cts in re sponse to cytokine s. 19 6 The gene ration of NO by iNOS is longlasting, in contrast to the cNOS and e NOS is oforms w here NO is ge ne rated in short bursts. 1 97 ,1 98 The me chanism of iNOS induction involves transcription of mRNA and nove l prote in synthesis and it takes se veral hours be fore NO is gene rated afte r the initiatin g signal. 1 99 It induce s a 100-fold highe r loc al conc entrations of NO than eNOS or c NOS and act as a antimicrobial de fenc e me chanism of the immune syste m. iNOS is not ex pre ssed in normal brains but ex pre ssion can be induce d in as troglial and mic roglial ce lls through viral infe ction or trauma. 2 00 ,2 01 It is mainly ex pre ssed under inflammatory c onditions, and after transie nt ischaemic periods. 2 02 - 20 5 Nitric oxide in neuropathology NO can be ne urotox ic under diffe re nt c irc umstanc es. The NO me diated ne uronal c ell de ath can be induce d by ove rex pre ssion of c NOS in ne urone s and astroglial ce lls or iNOS induc tion in glial ce lls, both pathw ays w ill be further discussed be low (se e Fig. 4 glutamate ne urotransmission le ading to ne urotoxicity has be en implic ated in Alzheimer's disease, Huntington's disease , amyotrophic lateral sclerosis (ALS), epilepsy and stroke . 20 6,2 07 Glumate ne urotoxicity is demonstrate d to be mediate d through NO production. After binding of glutamate w ith the NMDA subtype of glutamate re ce ptors, Ca 2+ ente rs the channel and binds to calmodulin, a cofactor for cNOS and stimulate s NOS activity w here by NO is produce d. 2 08 In addition, O -2 is produc ed w hich re sults in the formation of ONOOw hich subsequently leads to ne uronal de ath. 20 9 -21 1 Neurone s obtain ed from c NOS null transge nic mic e are markedly re sistant to is chae mic condition s, 21 2 in w hich the primary mechanism of damage is mediated by activation of the NMDA re ceptor and subse que nt formation of NO. This indic ates that cNOS is c apable of producing ne urotox ic amounts of NO. 21 3 iNOS in du c e d NO m e dia te d n e u ro to x icity The iNOS-me diate d re le as e of NO by astroc ytes and mic roglial c ells in the brain may be important in antimic robial or tumoricidal re sponse s to inflammatory signals. 1 97 In acute CNS inflammatory c onditions like rabies, herpes simplex , Borna, and lymphoc ytic choriome ningitis virus, iNOS is ex p re sse d 2 00 ,2 1 4 - 21 7 as w e ll as in ex pe rimental pne umococc al meningitis and tox oplasmosis and in humans during enc ephalitis . 13 0 ,2 18 -22 0 iNOS mediated NO is considere d to me diate ne uronal and oligodendroc yte de gene ration under ne uropathologic al c onditions . 20 2,2 21 ,2 2 2 The role of iNOS in ne uropathology in gene ral is indirec t, mic roglial c ells and/or mac rophages be come activated either through direc t infec tion w ith virus or othe r pathoge ns, or by loc al c ytokine production and subse que ntly produc e iNOS w hich e ve ntually le ads to damage . Ex c essive amounts of NO produce d by glial ce ll are probably ne urotox ic . iNOS-derive d NO is one of the major sourc es of tox ic fre e radicals in the brain, sinc e its re action w ith the sup erox ide anion (O -2 ) leads to the formation of pe rox ynitrite anion (ONOO -) w hich is an ex tre mely pote nt ox idizing agent. 22 3 Pe rox ynitrite gene rate s DNA-single-strand breaks w ith subse que nt activation of the DNA re pair enzyme poly ADP ribosyltransfe rase (PARS). 2 24 Furthermore , NO and perox ynitrite have be e n show n to inhibit the mitochondrial re spiratory chain and disrupt normal c ellular iron homeostasis. 2 01 ,2 25 -22 7 Pe rox ynitrite and/or NO can te rminally damage ne urone s, le ading to cell death. 2 28 -23 2 Low le vels of or sustaine d ex posure to NO or pe rox ynitrite cause apoptosis, w here as sudden ex posure to high conc entrations of NO or pe rox ynitrite le ads to ne c rosis . 23 3 NO in Alz he im e r's dis e a s e a n d MS iNOS ex pre ssion is found in Alzheimer's dise as e 23 4,23 5 and in ex perimentally infe cte d brains of rats w ith various viral age nts. 20 0 NO has be e n imp lic ated in demyelination and destruc tion of oligode ndrocytes and subsequent demye lination, the proce ss found in MS 236 and in the primary animal model for MS, ex pe rimental alle rgic e nce phalomyelitis (EAE). 2 16 ,23 7 During EAE, iNOS mRNA is dete ctable be fore the onse t of the clinic al symptoms and the leve ls of prote in corre late w ith the se ve rity of the disease . 20 0 Furthe r e videnc e supporting a role for iNOS in the pathogene sis of MS is the finding that human iNOS prote in and mRNA is marke dly elevate d in the ac tive lesions in brains of MS patients. 23 8,2 39 In MS le sions, macrophage s app ear to produce iNOS and are NADPH-diaphorase positive after histoche mical staining. 24 0 In another study, NADPH-diaphorase ac tivity in MS lesions w as found in re active astroglial c ells 23 9 w hich w as late r show n to be c NOS. 24 0 The c ellular sourc e of iNOS mRNA ex pre ssion in brains of patie nts w ith MS has be en c onfirme d to be macrophage s/ mic roglial ce lls. 2 38 In EAE an inc re ase w as found in eNOS in blood vessels in the inf lamed lesions and inc re as e in iNOS in infiltratin g inflammatory ce lls. 21

NO in AIDS de m e n tia c o m p lex
iNOS-me diated NO produc tion has also be e n de scribe d to be involve d in acquire d immune de ficie ncy syndrome (AIDS)-re lated ne urop athology. iNOS p rote in and mRNA w as found in the CNS of patie nts w ith AIDS deme ntia c omplex (ADC), 24 6,24 7 and is ex pre sse d at higher levels than in brains of AIDS patients w ithout ne urological symptoms. The ce ll type s ex pre ssing iNOS how ever, re main unknow n. In these patie nts there is a cle ar c orrelation be tw een the se verity of the de mentia and iNOS ex pre ssion in the brain. In addition in brain tissue of simian immunodefic ie nc y virus (SIV) infec te d monke ys iNOS mRNA w as dete c te d. 24 8 The microglial c ells have be en postulated to be involve d in the pathoge ne sis of ADC be c ause the y are pre fere ntially infec te d by the virus. 4 3,2 49 -2 51 Neuropathologic al manife stations such as loss of c ortic al ne urone s, loss of synapse s and ne uronal apoptosis have the re fore be e n suggeste d to be me diated indire ctly by c ytokines like TNFa 2 52 , 25 3 and IL-1 25 4 -2 56 or by nitric ox ide . 257 -2 61 TNFa , IL-1 and iNOS have be e n de monstrate d in brain s of AIDS patie nts 2 47 ,2 5 6,2 62 -2 65 as w e ll as in ADC patie nts 2 59 ,2 6 6,2 67 (Vinc e nt e t a l., submitted). In cytome galovirus (CMV) infe cte d re tinas from AIDS patients iNOS immunore ac tivity and NADPH-diaphorase w e re found in (CMV)-infe cte d ce lls identified as Müller c ells and as trocyte s. 26 8

NO in pa rkin s o n 's dis e a s e
In Parkin son's dise as e, w hich is primarily characte rized by a loss of midbrain dop amine rgic ne urones, iNOS w as pre sent in glial c ells 2 69 in the mese cep halon probably in ac tivated mac rop hages. 269 In addition, NOS inhibitor s attenuate malonate-induc ed degene ration by NMDA re ce ptor ac tivation of the nigrostriata l pathw ay in rats 2 70 sugge sting a role for NO in ne urodegene ration. The inhibition of tyrosine hydroxylase re sulting in re duce d dopamine synthesis is triggere d by perox ynitrite , a re ac tion product of NO.

NO in bra in a ctiva tio n
During ischaemic brain damage e NOS is induce d in endothelial ce lls w hich then has be ne ficial effec ts by enhancing the vasodilation, furthe r incre asing blood flow in the pe ri-infarct are a. 2 72 ,27 3 In addition iNOS is induc ed leading to NO production, w hich le ads to ne uronal de ath afte r c ere bral is chaemia. 27 4 During postnatal brain development of rats, large numbe rs of NADPH-diaphorase p ositive ne urone s and NADPHdiaphorase positive ce lls w ith mac rophage morphology w e re obse rved. The latte r are possibly involve d in developmental shaping of the brain, w hich inc ludes ce ll de ath and fagocytosis of ce llular de bris. 27 5 High levels of NADPH-diaphorase w e re found in e venly distributed astroglial ce lls in are as surrounding a me chanic al le sion in the brain. Within the le sion the NADPH-diaphorase positive cells most probably w ere macrophage s. 27 6 Thus, although iNOS and NADPH-diap horase ac tivity can be induce d in astroc ytes and mic roglial ce lls through viral infe ction or trauma most of the studie s have re vealed iNOS immunore ac tivity and iNOS mRNA in the brain in infiltratin g mac rophages or mic roglial cells. 2 10 In gene ral, ex posure of brain c ells to signals, such as mic robial produc ts, viruses, glutamate or ye t unknow n signals in dise as es like MS, Alzhe imer's and Parkinson's disease le ads to the se cre tion of inflammatory cytokines that induc e eithe r de n o vo synthesis of iNOS by glial c ells or cNOS in as troglial c ells or ne urons as has be en demonstrate d in in v itro studie s. This NO may be ne urotox ic and may subse quently lead to ne uropathology.

Production of iNOS in vitro
To answ er some more fundame ntal questions re garding iNOS production and re gulation in v itro studie s are w idely used. Sources, induction mechanism and inte rvention of iNOS p roduction are studied in de tail in various glial c ell cultures. In addition, a model has be e n developed that allow s studie s of the possible ne urotox ic e ffec ts of glial NO, on culture d ne urons. These in vitro studie s have le d to ne w insights in the functions of glial ce lls in normal brain and in ne uropathology.
Several te chniques have be en desc ribed for isolation of rat murine or human brain microglial c ells, astroglial c ells and maintenance of these ce lls in v itro . 1 18,2 4 0,2 77 -2 82 The microglial cells and astroglial ce lls are is olated from mix e d glial c ulture s in most instanc es or from disrupte d adult or ne onatal tissue. 5 3,1 2 7,1 71 ,2 7 9,2 8 3 - 2 85 In tissue c ulture , the ameboid and ramifie d microglial c ell can be ide ntified, most probably corre sponding to its morphological dive rsity in the adult brain. 28 6 The astroglial c ell c ulture s c an be contaminate d w ith microglial ce lls, since the purific ation of as troglial cells is a rather de lic ate te chnique and microglial c ells re main a significant contaminant. 5 6,1 61 ,2 87 Many studies show ing production of, for ex amp le , cytokines like IL-1 or TNFa by astroglial ce lls in v itro have to be interpre te d w ith care. In v itro studie s also provide a ve ry useful te chnique to study both the functions of glial ce lls and of ne urone s as w ell as the interac tions be tw een these ce ll types. There fore te chniques for se le ctive isolation, co-culturing, labeling and stimulation of mic roglial c ells and astroglial cells allow inve stigators to study some fundamental questions in ne uro-immunology.
Glia l c e ll-d e rive d iNOS iNOS ex pre ssion by glial ce lls has be e n studied in in v itro syste ms of highly purified microglial ce ll and astroglial ce ll c ultures and in mix e d c ulture s containing both c ell types from human or rode nt brain. Follow ing incubations w ith various stimuli, both mic roglial and as troglial c ells have be en demonstrate d to produc e nitrate, one of the e nd-p roduc ts of nitric ox ide ox idation. 20 3 iNOS has be en identified in rodent astroc ytes and mic roglial cells in re sponse to IL-1b , LPS or Grampositive bac te rial products. 18 5,2 59 ,2 88 ,2 8 9 IFNg induc e s NO in microglial ce lls and macrophage s but not in astroglial ce lls, 20 3,2 21 ,2 9 0 -29 2 w hile synergis m of IFNg and IL-1b or TNFa induc e signific ant levels of nitrite in rode nt and human astroglial c ells. 2 03,2 3 9,2 50 ,2 93,2 9 4 LPS induction of iNOS re quire d CD14 ex pre ssion on glial c ells. 2 95 As ye t it is not cle ar w he ther cytokines activate ge ne ex pre ssion via one or multiple pathw ays. Ex pe riments w ith p horbol este rs, w hich induc e iNOS, suggest that protein kinase C may be involve d in the induction p roc ess in microglial c ells and astrocyte s. 2 96 Agents like IFNg and LPS are more e ffec tive induc ers of iNOS in rodent than in human mic roglial ce lls, 19 8,2 50 ,2 8 8,2 9 7 w hich there fore appe ars to be spe cie s-de pende nt as described for NO production by re tinal pigme nt e pithe lial ce lls. 298 Rec ently, some studies did how e ve r re ve al NO and iNOS mRNA production in human ramified mic roglial c ells upon LPS or TNFa stimulation . 267 ,2 99 HIV or the HIV type I coat prote ins gp120 or gp41 induc e iNOS in c ulture d mic roglial ce lls, monocytes or macrophages. 9,2 46 ,2 57 ,2 5 8, 2 66 In human fetal glial ce lls, comparable amounts of NO w e re induc ed by gp120, gp41 and the proinflammatory c ytokine s IFNg and IL-1b . 9 HIV-infec te d brain mononucle ar mac rophage sec re te NO and O -2 , 2 66 e spe cially after immune activation and TNFa furthe r incre ase s NO production. In MS and EAE, macrophages isolated from lesion are as produc ed significant amounts of nitrite and w ere show n to be iNOS positive w ithout any furthe r stimulation. 24 0,3 00 b -amyloid, the major c ompone nt of the se nile plaques in Alzhe imer's dise ase , c ause s a significant inc re as e in NO by microglial ce lls and not in astroglial ce lls. The NO produc tion induce d by the b -amyloid w as inc re ased by IFNg 3 01 ,30 2 or phorbol-myristateacetate (PMA) challe nge. 1 68 Studie s that have atte mpte d to c ompare rat mic roglial c ell and astrocyte NO production have conclude d that mic roglial ce lls produce more NO on a per ce ll basis than astroc ytes. 20 2,2 22 ,2 9 0 This has le d to the sugge stion that activated mic roglial cells rather than astrocyte s are the principal source of re active nitrogen interme diate s in the CNS, and that the NO produce d by astroglial ce lls might be be ne fic ial, w here as mic roglial-derived NO might be involved in ne urotox ic ity. 18 5 Nitric o x ide m e dia te d n e u ro to xicity in v itro In vitro c o-c ulture s of glial c ells w ith ne urone s have proven to be a valuable tool in the ide ntific ation of NO as a ne urotox in and the ce llular source s of NO. Ex ogenous NO gene rate d from NO donors have be en show n to kill ne urones in v itro . 22 4 Cytokine-activate d murine microglial ce lls and astroglial ce lls appare ntly gene rate substantial amounts of NO that kill ne urones in v itro , 20 2,22 2 ,2 59 ,2 8 8,3 03 ,3 04 since inhibition of e ndogenously forme d NO by spec ific NOS inhibitors blocks this microglia-or as troc yte -mediated ne urotoxicity. 2 02 ,2 60,3 0 5 For ex ample, factors like LPS, gp41 and b -amyloid c an indirec tly kill ne urone s in mix e d cultures w ith glial ce lls, w hich is abrogate d by NOSinhibitor s. 24 6,3 01 LPS or cytokine ac tivated glial c ells stimulate the produc tion of ne urotox ins, e.g. NO, be c ause LPS and c ytokines do not direc tly influenc e the viability of purified ne urone s. 30 7 The se studies clearly illustrate the indire ct mechanism by w hich ne urone s are thought to be killed. 30 6 The glial c ells produce large amounts of NO by iNOS ac tivity w hich c an form pe rox ynitrite w hich is tox ic to ne urone s as pre viously described. In addition, glutamate ne urotox icity is also me diate d by NO in primary ne uronal cultures. 3 03 Pe rox ynitrite, NO and NMDA can damage ne urones in v itro le ading to ne crotic or apoptotic c ell de ath pe nding on the conc entration and duration of the ex p osure. 2 29 In addition, oligode ndroc ytes are also be ing kille d by an NO depe ndent me chanism by ameboid mic roglial ce lls in v itro , suggesting that iNOS ex pre ssion by invading and intrinsic brain cells p lay a role in lesion formation in multiple sclerosis. 2 21 Although glial ce lls produc e various ne urotox ins i.e. TNFa , glutamate and PAF, production of NO appears to play a ke y role in diffe re nt ne urotox ic pathw ays since inhibition of iNOS spare s the ne urone s. 2 60 ,3 05 ,3 0 8 These in v itro findings suggest an important role for glial iNOS derived NO in the pathophysiology of CNS dise as es.

Regulation of NO Production
Sinc e NO is show n to play an important role in ne urotox ic ity, inhibition of NO c ould be a possible route of interve ntion in the pre vention of ne uropathogene sis. Ex perime ntally ofte n used inhibitors of NOS p roduction are synthetic arginine analogue s like N G -mono-me thylarginine (NMMA), Nw -nitro-L-arginine methyl este r (L-NAME), aminoguanidine and N G -nitroarginine. 2 39 ,2 58 ,3 0 9 The y are often used in in vitro studie s to c ertify the L-arginine depe nde nt origin of NO.
Interve ntion of NO synthesis in vivo by using NOS spe cific inhibitors has be e n described in EAE using differe nt arginine analogues e .g. aminoguanidine, L-NMMA and L-NAME. 24 1,3 0 0,3 10 ,3 1 1 In these ex perime nts, the effe cts of the se inhibitor s on the clinical sc ore of EAE w ere not c onclusive. The aspe cific ity of these inhibitors for the subtype s of NOS, and the re by effects on eNOS and c NOS leading to e.g. vasc ular changes, may ex plain these differe nt re sults. The se arch for pharmac ological tools that selec tively inhibit iNOS, e NOS or c NOS is currently ge tting much atte ntion and thus far has yie lded some agents. The agent 7-nitroindazole is a selec tive c NOS inhibitor and N G -nitro-L-arginine show s a p re fere nc e for e NOS and cNOS over iNOS, w here as L-N 6 -(1-iminoethyl)lysine is se le ctive for iNOS over cNOS. 3 12 Intere stingly, NO can also ac t as a ne gative fee dback signal on iNOS ac tivity, by inhibiting the transcriptional induction of NOS, indicating a self-re gulatory me chanis m. 31 3,31 4 In cultures of rat astroglial cells nore pine phrine and dex amethas one have be en show n to suppre ss iNOS induction. 2 57 ,2 88 ,2 8 9, 3 15 In addition, prostanoids are involve d in the re gulation of iNOS sinc e prostaglandin E 2 but also cycloox yge nase inhibitor s suppre ss iNOS ex pre ssion in LPS activate d rat microglial ce lls. 3 16 In addition se ve ral endogenously produce d c ytokine s have be e n show n to inhibit NO produc tion. TGFb , IL-4 and IL-10, are know n to inhibit NOS activity in monocyte s 9 ,2 21,2 6 6,3 05 ,3 17 -32 0 and syne rgistically suppre ss NO produc tion. 70 TGFb is an important modulator in the brain during de velopment 32 1 and TGFb has be en demonstrate d in brains of HIV patients, around brain tumours, in MS, Alzheimer's dise ase , brain ischae mia, peripheral ne rve transec tions and in se ve ral ex perime ntal lesions in animals. 32 2 -33 0

Transforming growth factor b (TGFb )
TGFb is a 25 kDa homodime ric prote in sec re te d by a varie ty of c ells as a late nt protein complex . 6 0 There are at le as t five distinc t gene products that c onstitute the TGFb family, TGFb 1 through TGFb 5 w hich show a high de gree (70 -80%) of amino acid sequenc e identity. The thre e highly homologous mammalian TGFb isoforms are TGFb 1, TGFb 2 and TGFb 3 w ith re latively high se que nce similarities but differe nc es in re c eptor binding affinitie s. 3 31 Thre e diffe re nt TGFb re c eptors have be e n ide ntified, type I to type III, w hich distributions are ubiquitous in various body tis sues.
In a varie ty of studies of microglial ce ll cultures, TGFb has be e n show n to be a pote nt immunosuppre ssive cytokine 3 32 and inhibits NO produc tion by mic roglial c ells. 33 3 TGFb inhibits iNOS ex pre ssion by decre as ing iNOS mRNA stability and inhibiting its translation and inc re asing iNOS prote in degradation, 3 34 re sulting in re duced NO production. 3 17 ,33 5 Not only NO but also O -2 is inhibited by TGFb 7 1 and there by the formation of the highly tox ic perox ynitrite is pre vented.
TGFb is a chemotactic age nt for monocyte s and macrophage s and is suggeste d to be important in the re c ruitment of circulating monoc ytes into brain tissue after damage. 33 6 TGFb has protec tive effects in differe nt ex perimental autoimmune disease s 33 7-33 9 w here as ne utralizing antib odies to TGFb 1 w ors en clinical seve rity. 3 40 ,3 41 During EAE, and afte r ce re bral trauma or hypox ic-ischae mic damage , TGFb ex pre ssion is inc re ased w he n ne urological symp toms are se vere , w hich might indic ate an inflammation limiting of TGFb in the re cove ry phase there by controlling the inflammatory re action. 3 42 -34 5 Astroglial and mic roglial c ells are know n to c onstitutiv ely produce TGFb in v ivo 61,32 4,3 25 and in v itro . 62 ,1 0 3 -1 05 ,3 46 , 3 4 7 The is oforms TGFb 1, TGFb 2 and TGFb 3 are produce d by as troglial c ells in v itro , w hile mic roglial c ells only produc e the TGFb 1 isoform. 6 0 TGFb production in microglial and astroglial c ells is inc re as ed afte r TNFa 3 48 or IL-1 1 04 ,34 9 or TGFb ex posure itself. 35 0 In co-cultures of as troglial c ells and microglial cells bioac tive TGFb w as found to inhibit iNOS ex pre ssion and the re by NO production by endotox in activate d mic roglial ce lls. The p re senc e of astroglial ce lls w as show n to be e sse ntial for the ac tivation of TGFb in these co-cultures of astroglial and microglial cells 6 2 (se e Fig. 5).

Regulation of TGFb activity
TGFb is produc ed by glial ce lls in a latent, inac tive form 6 0 and forms a complex w ith the late ncyassociated prote in (LAP). 35 1,3 52 Ac tivation of late nt TGFb consists of re leasing TGFb from the LAP, w hich occ urs after he at treatment, acidific ation, alkalization or prote olysis by plasmin. 3

TGFb TGFb
Plasminogen Plasmin tPA uPA PAÌ Latent' TGFb`Active' TGFb and a 68 kD tissue-type p lasminogen ac tivator (tPA), se cre te d as inactive pro-forms, w hich major substrate is plasminogen. uPA and tPA can cle ave plasminogen into plasmin w hich subsequently activate s late nt TGFb 35 4,355 (Fig. 6). These PAs are spe cific ally inhibite d by the plasminogen activator inhibitors (PAI), PAI-1, PAI-2 and PAI-3 (Table 1), 35 6,3 57 by formation of a tight complex w ith PAs. Plasmin has a broad range of substrates including fibrin, fibrone ctin, laminin and matrix me talloproteinases (MMP). The PAs and PAIs play an important role by fine re gulating the prote olytic de gradation of fibrin clots (fibrinolysis) in the circulation, me diate d by plasmin 35 4,35 8 and the re fore tPA is now use d for the tre atment of thrombotic stroke. Only re c ently functions for PA w e re found in the brain, and proteolysis of the ex trac ellular matrix (ECM) by MMP activation, amplified by PAs, has be en suggeste d. The bre akdow n of the ECM is thought to be involve d in brain development and ne urite outgrow th but also in ne uropathology like grow th and invasion of brain tumours, leukoc yte infiltration in MS and EAE, breakdow n of the BBB and ne rve demyelination. 31 1 Microglial ce lls secre te prote ase s such as elastase, uPA and secre te p lasminogen 35 9,3 60 w hich have a dire ct ne urotrophic effe cts on various type s of ne urone s. 36 1 Astroglial ce lls c an synthesize and se cre te both tPA and uPA as w ell as PAI-1, 1 78 ,3 6 2 -36 4 and tPA is involved in motor learning but also in Alzhe imer's disease and ne uronal de gene ration. 3 65 -36 7 PAs and PAIs have be en demonstrated in ce re brospinal fluid of patie nts w ith ne urologic al disease 3 68 ,3 69 and tPA ex pre ssion w as found 37 0 in MS lesions supporting a role of PA and PAI in ne uropathologic al proce sse s.
An important role of tPA and PAI and the re gulation of TGFb activity w as show n in a glial ce ll coculture. 17 8 tPA and PAI-1 produce d by astroglial c ells re gulate d the bioac tivity of TGFb , and there by indire ctly the production of NO by microglial ce lls. 1 78 There fore , w e postulate that tPA-mediated activation of TGFb plays an important role in ne urop rote ction.

Summary
In ne uropathologic al c onditions such as Alzheime r's disease, Parkinson's dise as e, AIDS de mentia complex and multiple scle rosis , ac tivation of microglial cells and astroglial c ells is e vident. Unde r these ne uropathologic al conditions c ellular damage in the brain is c onsidere d to arise indirec tly from c ytotox ic substanc es p roduc ed by ac tivated glial ce lls. One of these tox ins is NO w hich has be en demonstrate d to be produc ed during several ne uropathological c onditions. High NO le ve ls are produced by glial ce lls and ex e rt ne urotox ic effec ts. Astroglial c ells and microglial ce lls c ommunicate in various w ays to re duce NO production by microglial c ells w hich is essential to maintain homeostasis in the brain. The production of TGFb by glial c ells and its ac tivation by astroc ytederive d tPA re pre se nts one mechanism by w hich astroglia limit NO produc tion in the brain.    Transforming grow th fac tor be ta e x p res sion in reac tive spinal cord mic roglia and me ninge al inf lammatory c e lls during ex p e rime ntal