key: cord-1042155-a9lmfaho authors: Eddleston, M.; Mucke, L. title: Molecular profile of reactive astrocytes—Implications for their role in neurologic disease date: 1993-05-31 journal: Neuroscience DOI: 10.1016/0306-4522(93)90380-x sha: 0b03acadefb0d7a3a233f34c4837d4c15197ea5f doc_id: 1042155 cord_uid: a9lmfaho Abstract The central nervous system responds to diverse neurologic injuries with a vigorous activation of astrocytes. While this phenomenon is found in many different species, its function is obscure. Understanding the molecular profile characteristic of reactive astrocytes should help define their function. The purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial fibrillary acidic protein. In vivo, this increase in glial fibrillary acidic protein-positive cells reflects predominantly phenotypic changes of resident astroglia rather than migration or proliferation of such cells. Upon activation, astrocytes upmodulate the expression of a large number of molecules. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. For example, upregulation of proteases and protease inhibitors could help remodel the extracellular matrix, regulate the concentration of different proteins in the neuropil and clear up debris from degenerating cells. Cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood-central nervous system interface. Neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostasis within the central nervous system. Therefore, an impairment of astroglial performance has the potential to exacerbate neuronal dysfunction. Based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. Among them are the relative contribution of microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive astrocytes with techniques that can resolve fast physiologic processes. Differences between reactive astrocytes in vivo and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism. the molecular prothe characteristic of reactive astrocytes should help define their function. The purpose of this review is to provide a summary of molecules whose levels of expression differentiate activated from resting astrocytes and to use the molecular profile of reactive astrocytes as the basis for speculations on the functions of these cells. At present, reactive astrocytosis is defined primarily as an increase in the number and size of cells expressing glial Sbriliary acidic protein. Jn r&q this incmase in gliaI tibrillary acidic prolix-~tive cells reflects ~~orninan~y pbenotypic changes of resident astrnglia rather than migration or proliferation of such cells, Upon activation, astrocytes upmodulate the expression of a large number of malecules. From this molecular profile it becomes apparent that reactive astrocytes may benefit the injured nervous system by participating in diverse biological processes. For example, upregulation of proteases and protease i~i~to~ could help remodei the extrac&ular matrix, regulate tire concentration of different proteins in the neuropil and ciear up debris from degenerating &is. Cytokines are key mediators of immunity and inflammation and could play a critical role in the regulation of the blood+entral nervous system interface. Neurotrophic factors, transporter molecules and enzymes involved in the metabolism of excitotoxic amino acids or in the antioxidant pathway may help protect neurons and other brain cells by controlling neurotoxin levels and contributing to homeostssis within the central nervous system. Therefore, an impairment of astroglial performance has the potentia1 FO exacerbate neuronai dysfunction. Based on the synopsis of studies presented, a number of issues become apparent that deserve a more extensive analysis. Among them are the relative contribution OF microglia and astrocytes to early wound repair, the characterization of astroglial subpopulations, the specificity of the astroglial response in different diseases as well as the analysis of reactive aatrocytes tith techniques that can resolve fast physiologic processes. DiEerences between reactive astrocytes in E&O and primary astrocytes in culture are discussed and underline the need for the development and exploitation of models that will allow the analysis of reactive astrocytes in the intact organism. 1, I~~DU~I~N physiologic and pathologic processes." One of the most remarkable ~ha~~~~s~~ of astrocytes is their Astrocytes make up a substantial proportion af vigorous response to diverse neurologic insults, a the CNS and participate in a variety of important feature that is well conserved across a variety of different species. The astroglial response (see section 2) *To whom correspondence should be addressed. occurs rapidly and can brz de&&d within one hour Abbr&atiorts: see over. of a focal mechanical trauma.201 Prominent reactive 1.5 astrocytosis is seen in AIDS demerttk" a variq of other viral infections,'"' prion-associated spongiform en~phaIopat~ies~ Rii in~ammatory dcmyel~na~~ng disease,"'.'53 acute traumatic brain injury,'" and such neurodegenerative diseases as Alzheimcr's disease." '.hh The prominence of astroglial reactions in various diseases, the rapidity of the astroglial response and the evolutionary conservation of reactive astrocytosis indicate that reactive astrocytes fulfill important functions for the CNS. Yet, the exact rofe reactive Abbrsviahzs: ACT, antichymotrypsin: AD, Alzheimer's disease; ADC, AlDS dementia complex; ALS, amyotrophic lateral sclerosis; APP, amyloid /? protein precursor; @A4 (aaf-421, amyloid 13 protein (amino acids I-42); BDNF, brain-derived neuro~ophi~ factor; Ca" I, calcium ionophore; CA II, carbonic anhydrase II; CAD, carbamyl phosphate synthetase II/aspartate transcarbamylase/dihydroorotase: CD, cluster designation; CJD, Creutzfeld-Jakob disease; CMV, cytomegalovirus; conA sup, supematant from concanavalin A-stimulated macrophages; &PM. central pontine myelinolysis: cpt-CAMP. g"(~-ebiorop~enyi thief adenosine 3'-5-cyclic monopbosphae; protein kinase C; PMA, phorbol-IZ-mvristate-1 Z-ace&e; PN, proteese nexin; PTBBS, peripheral type benzodiazepine binding site; SGP, sulphated glycoprotein: SSPE, subacute sclerosing pane?~~p~a~itis~ sub P, substance P; TGF, transforming growth factor; TIMP, tissue inhibitor of metailoprotease; 'US, TPA induced sequences; TMEV, Theiler's murine enccphalomyelitis virus; TNF, tumour necrosis factor; TPA, 12-O-tetradecannyl-phorbol-13-acetate; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminopn activator; trauma, focal mechanical or electrolytic destruction of CNS tissue; VW', vasoactive intestinal peptide: VLA, fi 1 integrin famity. astrocytes pIay in the injured C'NS has so l'al-rzn~~~ncd efusive. Assuming that the hiotugical funcrions of IWCtive astrocytes are reffected in the proteins the!, express, this review aims to further our undcrstanding of these cells by providing a synopsis ot' rcccnt studies examining the molecular profile of activated astrocytes. The CNS responds to neural injuries with an increase in the number and size of cells expressing glial fibrillary acidic protein (GFAP), a phenomenon generally referred to as reactjve astrocytosis. GFAP is an intermediate filament cytoskefetal protein expressed primarily by astrogliaz9 and represents the prototypic marker of astroglial activation.z8s60 However, despite its prominent upmodulation in response to diverse injuries, the precise function of the GFAP molecule remains uncIear. Suppression of GFAP expression in gliaf ceil lines with antisense mRNAs suggests that GFAP may be necessary for tbe formation of stable glial processes in response to neuronai signals."" It will be interesting to assess the functional role of GFAP in vivo by ablating GFAP in experimental animals with the help of homologous recombination, expression of anti-sense mRNAs or ribozymes. It sboutd be noted that it has not yet been established iF an increased level of GFAP expression and/or turnover is, in fact, a reliable indicator of astroglial activity in general. For example, in normal rodent brains, astrocytes of the glial limitans and the hippocampal formation show higher levels of GFAP mRNA and GFAP immunostainin~ than astrocytes of other brain regions.".'Si.i5".1h7.?"1 This raises the question whether these heterogenous levels of GFAP expression reflect particular functional demands placed upon specific astroglial subpopulations and whether they correlate with a general increase in the functional activity!ltl~tabolism of the strong& GFAF-positive cells. It should also be noted that using increased GFAP expression as the basis for the definition of astroglial activation will exclude any subpopulation of astrocytes that responds to neural injury without GFAP expression. Pending further experimental evaluation of these issues we have considered the induction of GFAP expression to be the main indicator of astroglial activation. 'The origin of the increased number of GFAPexpressing cells that appear in response to ncurologic insults has been the subject of intense discussion over the last decade, Specific&y, the debate has focused on the question of whether reactive astrocytosis represents primarily the prol~ferat~on/migration of GFAP-positive cells or the phenotypic change of local astrocytes. Studies using double-labeling with GFAP antibodies and bromodeoxyuridiae or tritiated thymidine to identify dividing astrocytes have shown that, ar least in acute lesions, mitotic division (pro-for the majority of GFAP-positive cells that appear in response to the injury (for review, see Ref. 211) . Furthermore, we have been unable to find convincing in vivo evidence that mature GFAP-positive astrocytes of adult brains are able to migrate effectively. Hence, it is likely that the appearance of GFAPpositive astrocytes in regions of acute neural injury represents primarily a change in the phenotype of resident astroglia. It can, however, not be excluded that astroglial proliferation contributes more significantly to chronic astrocytosis. In many instances, the phenotypic changes seen in reactive astrocytes may reflect a substantial increase in astroglial metabolism and protein synthesis, consistent with a "healthy" cellular hypertrophy in response to increased physiologic demands. In other situations, however, astroglial swelling may result from pathologic processes that afflict the astrocyte itself (for review, see Ref. 210). The current literature on reactive astrocytosis is extensive. We have attempted a comprehensive review of this subject using rigid selection criteria to produce a practical synthesis that will be easily amenable to consultation. To construct a list of molecules expressed by activated astrocytes we have included information drawn from two types of studies. The first are studies carried out in vivo where the expression of a particular molecule or its mRNA was co-localized to reactive astrocytes by immunochemical staining or in situ hybridization. For inclusion into the table clear evidence for astroglial expression was required, for example co-labelling with GFAP or demonstration of electron-microscopic features typical of astrocytes. This should ensure that the molecules in question were indeed found in astrocytes rather than in other injury-responsive CNS cells, in particular microglia. The combination of immunostaining with in situ hybridization can also help differentiate between accumulation in astrocytes of molecules actually synthesized by these cells and those produced elsewhere and subsequently taken up by the astrocyte. Many interesting leads on the induction of potential astroglial molecules, particularly enzymes, have come from studies on bulk brain extracts. However, because these studies usually do not provide direct proof that astrocytes form the main cellular source of the identified molecules in the patholo~~lly altered CNS, they have not been included in this review. The second class of information comes from in vitro studies. Since the isolation of enriched astrocyte cultures by McCarthy and de Vellis192 and subsequent refinements, a great deal of experimental work on astrocytes has been carried out in vitro. Experiments indicating the upregulation of a molecule by a certain factor in vitro may offer clues as to what happens during reactive astrocytosis in the CNS, especially if this factor is known to occur in pathological conditions. However, while tissue culture studies often provide important leads they can also sometimes be misleading. Therefore, because so much of our current knowledge on astrocytes is based on in vitro studies we would like to address a Few caveats that should be kept in mind when considering the molecular profile of cultured astrocytes. A major consideration is the imperfect purity of primary astrocyte cultures because current techniques for purifying astrocytes usually produce cultures of 90-99% purity. Contamination with microglia is particularly problematic because these cells also respond to neural injuries and secrete a number of biologically active molecules such as cytokines. At present, the most definitive assay for determining the cell source of most molecules is the combination of immunostaining with in situ hybridisation but this has been carried out only rarely (for an example, see Ref. 287). For inclusion into Table 1 , we have favored studies that have addressed the issue of culture purity. The adult CNS is characterized by the close interaction of many different cell types both through actual cell contact and secretion of factors. Thus a further problem is that cells in nearly pure primary culture have been released from these interactions. This point is illustrated by the fact that astrocytes in tissue culture have different morphologies depending on whether they are cultured alone or with other neural cells. Cultured alone, they bear few processes, however when co-cultured with neurons they develop multiple processes. 'lo The physiolo~c behavior of astrocytes is also dependent on the presence of other neural cells. Cocultivation of astrocytes with neurons induces calcium channel activity in astrocytes which is undetectable in pure astrocyte cultures or in astrocytes co-cultured with oligodendrocytess6 Many protocols for the establishment of primary astrocyte cultures include an early exposure of the cells to relatively high concentrations of serum. This represents a major difference from the situation in vivo where astrocytes are shielded from blood-derived factors by the blood-brain barrier. In essence there are numerous variables in culture conditions that could dramatically influence the molecular profile of astrocytes in vitro and alter the astroglial responsiveness to further stimulation. Cloned lines of immortalized glial cells such as the rat glioma cell line C6 can circumvent the problem of culture impurity and have yielded an enormous amount of interesting data. However, they differ from astrocytes in viva in many respects, even more so than primary astrocytes. As an example, astrocytes of the adult CNS have only a limited proliferative potential'72~2'1 and this is reflected to some extent in primary culture. In contrast, immortalized cell lines often proliferate vigorously having been released from many controlling influences, including in some cases contact inhibition. Therefore, findings obtained with immortalized glial cell lines have not been included in Table 1 IO,11 IO,11 10 10 9 9 9 9 10,ll IO,11 10 9 9 9 9 10,ll IO,11 10 IO,11 10,ll 10 10 9 9 9 9 10 10 10 10 9 The assignment of molecules to a specific functional category was introduced to facilitate consultation of the table. Note, however, that this assignment is somewhat arbitrary as a number of molecules can exist in different forms or fulfill functions in different categories. For example, there is evidence that components of the amyloid fi protein precursor (APP) could function as a protease inhibitor2'4s273 or as a serine proteasg2' while the structure of the whole precursor molecule resembles that of a cell-surface receptor '38. In addition, it seems likely that other functions will be identified for many of the above molecules, some of which may be more relevant to the CNS than those they are currently assigned. Because GFAP is a well established marker for reactive astrocytes and colocalization with GFAP was required for inclusion into the table this molecule has not been listed. Separation of inducer molecules/conditions by commas indicates that each inducer was effective when tested in isolation, whereas a plus sign indicates that synergistic effects were observed when both inducers were combined. References in square brackets [] contradict the previously quoted reference. For definitions, see abbreviations list. The transition of astrocytes from the resting to the activated state is associated with the expression of new molecules not normally detectable in quiescent astroglia as well as with the upmodulation of factors that are found in resting astrocytes at lower levels. Table 1 lists a number of molecules whose expression in astrocytes increases upon astroglial stimulation and, hence, may provide a molecular profile of reactive astrocytosis. From this table, it appears that reactive astrocytes are equipped with a large armamentarium of molecules that allows them to participate in many important biologic functions. In the subsequent sections we will speculate how the expression of specific groups of molecules could relate to the function of reactive astrocytes. As outlined above astrocytes undergo dramatic changes upon activation which are likely to have functional consequences. It remains, however, controversial if the induced changes are generally beneficial or detrimental in nature (reviewed in Ref. 231). On one hand, it is conceivable that the increase in cytoskeletal proteins within reactive astrocytes may assist wound repair by stabilizing the tissue surrounding neural injuries. The glial scar formed by reactive astrocytes may also help to wall off areas of tissue necrosis, excluding non-neural cells from the CNS parenchyma and appears to fill in the space that results from neuronal 10~s.~~ On the other hand, it has been suggested that the glial scar may form a barrier that could hinder regenerative processes such as neurite outgrowth.2~~232 Central neurons do not regenerate effectively after injury. The studies of Aguayo and colleagues indicate that this is due not to an intrinsic inability of these neurons to regenerate but to the environment present within the CNS.* Electron-microscopic analysis of regenerating axons revealed that arrest of axonal growth in the CNS occurs in the immediate vicinity of reactive astrocytes. 175 This observation together with the finding that reactive astrocytes in vivo express molecules which inhibit neurite extension in vitro'93 suggests that astrocytes can actively inhibit regeneration. While it is difficult to prove that dense gliotic scars do not mechanically block axonal growth, in vitro evidence suggests that astrocytes themselves are not necessarily inhibitory to regeneration (reviewed in Refs 111, 182) . Furthermore., reactive astrocytes do not prevent PC12 cells from extending neurites over glial scars in optic nerve explants.'j3 Most conclusively, the in vivo experiments of Gage and Kawaja showed that in the presence of NGF (produced by transplanted fibroblasts), reactive astrocytes could, in fact, provide a substrate for the growth of sympathetic neurites.'" These findings demonstrate that, at least in certain experimental situations, astrocytes do not inhibit but may even promote regeneration. A role for reactive astrocytes in regeneration and tissue repair is also supported by their molecular profile (see Table 1 ) which suggests both a production of, and interaction with, the extracellular matrix. In vivo astrocytes express extracellular matrix molecules such as laminin, chondroitin-6-sulphate proteoglycan and glial hyaluronate adhesion protein, a hyaluronate binding protein. In vitro, they are also able to secrete glycosaminoglycans.*J35 Reactive astrocytes may interact with extracellular matrix and other CNS cells via adhesion molecules such as embryonic neural cell adhesion molecule and cytotactin/tenascin. Transforming growth factor (TGF)-/l I has been shown to be increased in reactive astrocytes after CNS stab wounds."' Logan and colleagues proposed that astroglial secretion of TGF-fl 1 may attract fibroblasts into the lesion site, regulate their deposition of extracellular matrix proteins and synthesis of degradative enzymes, and play a role in controlling angiogenesis in the scar. Hence, astrocytes may be important in controlling the deposition of scar tissue after injury and its vascularization."' The production of proteases and protease inhibitors might allow astrocytes to further remodel the extracellular matrix at sites of neural injury and to clear up the debris of degenerating cells. While the activity of these molecules would thus assist in wound repair it is also conceivable that astroglial proteases or protease inhibitors have detrimental effects in certain pathologic conditions. The production of calcium activated proteases by reactive astrocytes has, for example, been implicated in the degeneration of neurons after ischemia, and in the production of the amyloid /3 protein,'" a protein that accumulates abnormally in the brains of patients with Alzheimer's disease. Destruction or degeneration of white matter tracts in the CNS leads to the release of large quantities of myelin lipids. Apolipoprotein E (apoE) is a major constituent of both low-and high-density lipoproteins and plays an important role in lipid transport and metabolism. Within the CNS apoE is constitutively produced by astrocytes,"~202~'5' and the astroglial expression of apoE has been found to be upmodulated during reactive astrocytosis."' Astrocyte-derived apoE may help deliver lipids to other CNS cells for membrane biosynthesis and facilitate the removal of cholesterol into the periphery. Consistent with the latter possibility is the increase in plasma apoE levels observed during the active phase of experimental allergic encephalomyelitis (EAE).'"' a demyelinating disease of the CNS. One of the major functions proposed for reactive astrocytes is the initiation of immune responses within the CNS (e.g., see Ref. 112 ). When treated with factors such as interferon-y, astrocytes in vitro are induced to express molecules involved in immune responses, for example major histocompatibility complex (MHC) antigens and adhesion molecules such as intercellular adhesion molecule 1. Cultured astrocytes are able to present antigens to MHC class I and to MHC class II restricted T lymphocytes80~8'~'74,26' and to produce many different cytokines. In addition, a number of in vivo immunohistochemical studies have reported the expression of MHC molecules on small numbers of reactive astrocytes in different pathologic conditions (see Table I ). Taken together, these findings support speculations that (i) antigen presentation by MHC expressing astrocytes and astroglial production of cytokines might play a crucial role in CNS-immune interactions; and that (ii) interactions are mediated primarily by microglia rather than by astrocytes.".'*' '23~'4'~'ch~'5X~'RR These studies indicate that astrocytes probably do not function as the main antigen presenting cells in the CNS and argue against a major role for astrocytcs in the initiation of immune-mediated neurologic diseases. However, as outlined below, astrocytes may still have important regulatory effects on inflammatory and immune responses directed at the CNS. The interaction of the CNS with blood-borne factors and cells is of paramount importance in the pathogenesis of a number of neurologic diseases. This interaction is controlled, in part. by the blood-brain barrier which is formed by the unique properties of the CNS endothelial cells. Astrocytes are in intimate contact with these cells by their endfeet processes**' and several lines of evidence suggest that they may participate in the control of the blood-CNS interface. Astrocytes could influence the entry of hematogenous cells into the CNS as well as their intraparenchymal activity through the secretion of cytokines. As indicated in Table 1 , astrocytes appear to produce a large number of cytokines and inflammatory mediators in vitro. Unfortunately. in Gw confirmation of these findings is lacking in most cases and the possibility of microglial contamination of astrocyte cultures has not always been addressed rigorously. However, the few in vivo studies that are available support the postulate that astroglial cytokine production may be involved in the pathogenesis of viral and immune mediated neurologic diseases. For example, Wahl and colleagues*" have shown that reactive astrocytes in HIV-1 infected brains express TGFP and speculate that this cytokine enhances the recruitment of HIV-l -infected monocytic cells. Hence, the astroglial TGF,0 production could both contribute to the inflammatory changes seen in HIV-I associated encephalomyelitis and also increase the spread of cell-borne virus in(to) the CNS. It should be noted in this context, however, that many cytokines appear to fulfill a multitude of functions (for review see Ref. 26.5 ) and that their effects in the intact adult CNS are only now beginning to be defined." It is, therefore, perhaps not too surprising that the effects of cytokines in specific neurologic diseases have been difficult to predict.'0.'9.'74.'4h Proteases and protease inhibitors could be used by astrocytes to regulate the concentration of a variety of proteins in the parenchyma, including cytokines and proteases derived from the blood or from other brain cells. Such a role has recently been suggested for protease nexin 1,5'*'27 a protease inhibitor found to be increased in reactive astrocytes.'*' In vitro data indicate that protease-protease inhibitor complexes can induce the synthesis of acute phase proteins in response to injury'q~'s2 and stimulate the directed migration of neutrophils. Is Because reactive astrocytes express both cathepsin G-like protease and alphalantichymotrypsin-like protease inhibitor activities (Abraham et al., unpublished observations) such complexes may form around reactive astrocytes where they would directly or indirectly increase the release of cytokines and acute phase proteins from astrocytes, endothelial cells, microglia or blood derived In head trauma and intracerebral hemorrhage the blood-CNS interface is acutely disrupted. This disruption causes red blood cells to extravasate, lyse and release iron-containing compounds into the CNS. Consequences of such lesions include focal encephalomalacia, hemosiderin deposition and occasionally the development of recurrent seizures. Studies in experimental animals suggest that some of the clinical sequelae of brain trauma are related to the induction of free radicals by the iron moieties within extravasated blood, and the subsequent peroxidation of lipids. 282 The expression of transfer&, which mobilizes and transports iron, and its receptor in reactive astrocytes 55395*2'5 suggests that these cells may help diminish excess iron loads around sites of tissue injury. The blood-brain barrier shields the CNS from toxic metals present within the blood. However, in a number of locations the blood-brain barrier is leaky. *' Surrounding these sites one finds a class of GFAP-positive cells termed Gomori astrocytes (reviewed in Ref. 245) which may have an important role in controlling metal toxicity. These cells increase in number after irradiation256 and accumulate silver, mercury and lead after systemic administration of these compounds. 245 Gomori astrocytes express metallothionein,289 a protein which can bind to heavy metals such as cadmium and mercury, detoxifying them in the process. The protein is inducible by heavy metals in various tissues and there is some evidence that this occurs in astrocytes after cadmium administration.*08 Tissue factor or tissue thromboplastin is a transmembrane glycoprotein that functions as the initiator of the coagulation protease cascade. In the brain tissue factor is expressed predominantly in astrocytes.'*" In view of the apposition of astroglial endfeet with CNS endothelial cells (see above), tissue factor could help astrocytes form a "hemostatic envelope" around the vascular system of the CNS. The upregulation of tissue factor expression by reactive astrocytes in nonhemorrhagic conditions such as scrapie suggests that tissue factor may fulfill additional functions within the CNS. While it has long been realized that astrocytes secrete factors that promote the growth and prolong the survival of neurons in explant culture,16 so far only a limited number of astroglial molecules that exert trophic effects on neurons have been identified. However, it seems likely that this small group represents the tip of the iceberg. As outlined below some astroglial neurotrophic factors may act directly on neurons whereas others could benefit neurons indirectly through the support of other CNS cells. Both nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) act as survival and neurite extension factors for some types of cultured neurons.'99~278~286 Astrocytes, in contrast to microglia, are ablear to secret NGF in vitro.*" After trauma, NGF levels are increased in both the optic nerve"' and the hippocampus,'60~28' and in a separate study, the cellular source of NGF was shown to be astrocytes. I4 Astrocytes also produce bFGF in vitro in response to various factors, and in Alzheimer's disease and lesioned brain bFGF has been localized to reactive astrocytes (see Table 1 ). Recent evidence from tissue culture studies suggests that growth factors such as NGF and bFGF are able to protect central neurons against hypoglycemic/excitotoxic insults by stabilizing neuronal calcium hemostasis.48'90'9' Reactive astrocytes produce insulin-like growth factor-1 (IGF-1) after ischemia.94J63 Because IGF-1 has neurotrophic effects,46.94 this astroglial response may help diminish neuronal loss. IGF-1 also stimulates oligodendrocyte development and myelination in vitro.m Work with mice transgenic for IGF-1 supports a similar role for the molecule in vivo.47a Consistent with the postulated role of IGF-1 in myelination, both IGF-1 and its receptor decrease to minimal levels in the adult brain.'3'8,33 Reactive astrocytes have recently been shown to express IGF-1 '49 concomitant with the expression of the IGF-I receptor by immature oligodendrocytes around the lesion.'49 This raises the possibility that reactive astrocytes play an important role in the remyelination of the adult CNS. However, reactive astrocytes expressing tumor necrosis factor 01 (TNFcr) have been identified in multiple sclerosis lesions.'28~248 While there is no direct evidence for a role of astrocyte-derived TNFa in demyelination in vivo, this cytokine has been shown to be toxic to oligodendrocytes in culture. 234,249 Consequently, it remains undecided at this point if the role of astrocytes in inflammatory demyelinating diseases is beneficial or detrimental. High concentrations of excitatory neurotransmitters are extremely toxic to neurons (reviewed in Ref. 52). Evidence is increasing that the neuronal death or impairment that follows acute neurologic insults (e.g. hypoxia/ischemia, mechanical trauma, prolonged seizures) may, in the large part, be mediated by an increase in the extracellular concentration of excitatory amino acids such as glutamate. A role for glutamate toxicity has also been proposed in more chronic neurologic diseases such as Alzheimer's disease,'47,'xY AIDS dementia (reviewed in Ref. 173) , sulfite oxidase deficiency, Guam amyotrophic lateral sclerosis and Huntington's disease (reviewed in Ref. 52). In the presence of high glutamate levels, removal of astrocytes from mixed cultures quickly leads to neuronal cell death.2"."8.260 In vitro studies suggest that amino acid transmitters may be removed from the extracellular space by astrocytic uptake mechanisms (reviewed in Refs 79, 113, 132) . Astrocytes also contain glutamine synthetase which converts glutamate to glutamine and helps detoxify ammonia in the CNS. This enzyme has been shown to be upmodulated in reactive astrocytes in pathologic conditions.4'.2"" Hence, it is possible that astrocytes participate in the removal of neurotoxins by both enhanced uptake and metabolic turnover. The recent cloning of the transporters for GABA and the amines, noradrenalin, serotonin and dopamine (for review see Refs 254, 272) should supply molecular tools that will help in understanding the role of reactive astrocytes in regulating other neurotransmitters. In a number of recent studies, Heyes and his colleagues have provided evidence that the NMDA receptor agonist quinolinic acid is involved in the pathogenesis of the neurologic dysfunction that can be associated with HIV-I infection and other inflammatory diseases of the nervous system.'"."' I20 Because the quinolinic acid metabolizing enzymes, 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase, have been localized to astrocytes in vivo, '50~'5' it is conceivable that the expression of these enzymes increases in astrocytes responding to inflammatory lesions. While an upmodulation of these enzymes in reactive astrocytes has apparently not yet been documented in the literature such an astroglial response could serve important protective functions in a variety of neurologic diseases. Free radicals form another group of chemicals that could be extremely toxic to the nervous system'02,'0' and the ability to eliminate or control these entities may be critical after neurologic insults such as cerebral hemorrhage. 282 While this issue does not appear to have been directly studied in reactive astrocytes, there is evidence that astrocytes may play a role in the antioxidant defense system. The biopigments biliverdin and bilirubin are potent antioxidants.258 They are synthesized by a pathway which is rate-limited by the heme oxygenase isozymes HO-1 and HO-2. HO-I is expressed by astrocytes in culture72 and induced in glial cells after heat shock to the rat brain.74 Apolipoprotein D, proposed also to be involved in the production of antioxidants, appears to be expressed by astrocytes in the normal CNS" and increases in the peripheral nervous system after injury.'" Antioxidant enzymes such as superoxide dismutase and catalase have been proposed to be induced in reactive astrocytes in Alzheimer's disease."" If the above studies are confirmed by double-labeling of reactive astrocytes it would be interesting to know if the antioxidant enzymes are induced solely to protect the astrocytes themselves or whether they are also secreted to influence the environment of other neural cells. In this review we have constructed a molecular profile of reactive astrocytes and drawn conclusions from this profile on the functions reactive astrocytes may fulfill in neurologic diseases. As a result we have hypothesized that activated astroglia may benefit the damaged nervous system by participating in several important biologic processes such as the regulation of neurotransmitter levels, the repair of the extracellular matrix, control of the blood-CNS interface, transport processes, and trophic support of other CNS cells. The detectability of specific molecules depends not only on their absolute levels but also on the sensitivity of the assays used, i.e. the inability to detect certain markers does not necessarily exclude their presence. Consequently, it cannot be excluded that "resting" astrocytes also fulfill some of the functions assigned to reactive astrocytes but at a lower level. We would like to emphasize that our extrapolation of the functions of reactive astrocytes from the molecules they express is speculative and based on current knowledge. It seems likely that other functions will be identified for many of these molecules, some of which may be more relevant to the CNS than the ones they are currently assigned. We also expect that the ongoing discovery of CNS-specific genes (see Ref. 198 for review) and the development of novel molecular probes/assays will significantly expand the molecular profile of reactive astrocytes. In the majority of CNS diseases clinical signs and symptoms are related most directly to an impairment of neuronal functions. While little evidence exists that the activity of reactive astrocytes is directly detrimental to the nervous system, it is conceivable that an impairment of astroglial performance could exacerbate neuronal dysfunction. This pathogenetic scenario may, for example, exist in hepatic encephalopathy (see Ref. 210 for review), scrapie in which prions appear to accumulate first in astrocytes" or in AIDS dementia where viral or macrophage-derived products could interfere with astroglial functions such as neurotrophic support and/or elimination of excitotoxins,36.37,49.173,228 An inspection of Table 1 reveals that reactive astrocytes express a number of molecules that are typically produced by hematogenous cells. This observation could reflect the evolutionary response of the CNS to two different types of selective pressures. neurophysiologic processes that occur within seconds. There appears to be a need for the CNS to restrict For example, the response of neurons to electrical the access of hematogenous cells as evidenced by the stimulation was shown to be accompanied by rapid blood-brain barrier and the delayed invasion of Ca*+ oscillations within astrocytes in hippocampal neutrophils and monocytes after injections of LPS slice preparations. 62 Astrocytes themselves are also into the brain parenchyma when compared with capable of responding to neurotransmitters (reviewed peripheral sites.' On the other hand, early stages of in Refs 19, 26) . Because of their close association with wound repair within the CNS may depend on the nodes of Ranvier,32*'% perinodal astrocytes may be fast action of those factors which are released into in a particularly suitable position to influence neuroperipheral wounds by hematogenous cells. Recent physiologic processes. It is possible that rapid evidence suggest that astrocytes are able to respond responses of astrocytes are of greater functional to neural injury with great rapidity.'"*'31s"' Therefore, importance in neurologic diseases than the molecular astrocytes may fulfill some of the functions that are changes that occur over hours or days. Yet, this type carried out by invading hematogenous cells during of response cannot be detected with conventional wound repair in peripheral sites. We would like to histopathologic methods. The application of novel emphasize at this point that the response of the CNS neurophysiologic and cell biologic techniques should to neurologic injury involves many cell types in allow a high chronologic and spatial resolution of addition to astrocytes and that the assignment of astroglial responses and is expected to substantially certain functions to astrocytes by no means excludes further our understanding of astroglial functions the participation of other cells. An assessment of the in health and disease. We suspect that this type of relative contributions of microglia and astrocytes to analysis will reveal "reactive astrocytosis" to be a early wound repair within the CNS should be a much more dynamic process than is currently particularly fruitful subject for future studies. conceptualized. Recent data indicate that subpopulations of astrocytes can be distinguished both at the molecular'7*'64,'83~'97 and functiona16',**' levels. In leukocyte research the development of molecular markers has revealed a great functional diversity among cells that appear morphologically very similar. It seems likely that ,future molecular studies will also reveal a functional heterogeneity of reactive astrocytes that far surpasses their morphologic differences. It will be particularly interesting to find out whether there are subpopulations of astrocytes that respond to some neurologic disease processes but not to others. In a similar vein, it needs to be determined whether diverse neurologic diseases provoke the expression of the same set of astroglial molecules or whether the astroglial response is specific, with different molecules being expressed by astrocytes responding to different neurologic insults. We would like to end this Commentary by pointing out the imbalance between in vitro and in vivo studies in astroghal research. Judged by the number of in vitro vs in vivo studies (see Table 1 ), much greater efforts appear to have been placed on the extensive analysis of astrocytes in culture than on the in vivo confirmation of existing in vitro findings. However, reactive astrocytes in the adult brain and primary astrocytes in ccl1 culture differ in many respects and results obtained in vitro and in vivo often do not overlap (see Table 1 and, for an example, Ref. 170) . It is, therefore, to be hoped that future research will complement the vigorous efforts made in cell culture systems with the development and exploitation of models that allow the analysis of reactive astrocytes in the intact organism. It should also be pointed out that the response of astrocytes to neurologic insults has so far been documented primarily by immunohistochemical staining and in situ hybridization. This methodologic approach provides a static image of the molecular profile of reactive astrocytes and does not allow the resolution of fast physiologic changes. Recent evidence suggests that astrocytes participate in central nervous system myelination in transgenic mice NGF and bFGF protect rat hippocampal and human cortical neurons against h~o~y~~c damage by stabilising calcium homeostasis Human immunodeficiency virus can productively infect cultured human glial cells &expression of glial fibrillary acidic protein and vimentin in the central and peripheral nervous systems of the twitcher mutant. Gfii I 11990) Protease Nexin-I Localization in the human brain suggests a protective role against extravasated serine proteases Glutamate neurotoxicity and diseases of the nervous system Tumor necrosis factor-alpha production by astrocytes. Induction by lipopoly~~ha~de, IFN-gamma, and IL-lbeta Characterization and differential distribution of the three major human protein kinase C isozymes (PKC alpha, PKC beta, and PKC gamma) of the central nervous system in normal and Alzheimer's disease brains A histochemical study of iron, transferrin, and ferritin in Alxheimer's diseased brains Neuronal modulation of calcium channel activity in cultured rat astrocytes Regulation and selective expression of Ly-6A/E, a lymphocyte activation molecule, in the central nervous system Glial cell-specific mechanisms of TGF-81 induction by IL-1 in cerebral cortex Transforming growth factor-beta 1 (TGF-beta I) expression and regulation in rat cortical astrocytes Heterogeneity of the glial fibrillary acidic protein in gliosed human brain Filament proteins in rat optic nerves undergoing Wallerian degeneration: localization of vimentin, the fibroblastic 100-A filament protein, in normal and reactive astrocytes Neuronal activity triggers calcium waves in hippocampal astrocyte networks Macrophages can modify the non~~issive nature of the adult mammalian central nervous system Castration enhances expression of glial fibrillary acidic protein and sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat Subacute encephalomyelitis of AIDS and its relation to HTLV-III infection General and dramatic glial reaction in Alzheimer brains Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurons Astrocytes and microglia in human brain share an epitope recognized by a B-lymphocyte-specific monoclonal antibody (LN-1) Scrapie-associated priori protein accumulates in astrocytes during scrapie infection Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of apoli~protein E and cathepsin D in astrocytes Regulation of heat shock protein synthesis in ral astrocytes Heme oxygenase is a heat shock protem and PEST protein in rat astroglial cells Astrocytes are the primary source of tissue factor in the murine central nervous system-a role for astrocytes in cerebral hemostasis Selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion Normal and heat-induced patterns of expression of heme oxygenase-I (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein Astrocyte lineage. 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Part II: unlike H-ZK-dependent cytotoxic T cells, H-2Ia-restricted T cells are only stimulated in the presence of interferon gamma Astrocytes present myelin basic protein to encephalitogenic T-cell lines Production of prostaglandin E and an interleukin-I like factor by cultured astrocytes and C6 glioma cells Expression of class II major histocompatibiiity antigens on reactive astrocytes and endothelial cells within the gliosis surrounding metastases and abscesses On the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases The induction of inter~llular adhesion molecule 1 @CAM-l) expression on human fetal astrocytes by interferon-gamma, tumor necrosis factor alpha, lymphotoxin, and interleukin-1: relevance to intracerebral antigen presentation Astrocytes and intracerebral immune responses Interleukin-1 beta and tumor necrosis factor-alpha synergistically stimulate nerve growth factor (NGF) release from cultured rat astrocytes Subacute spongiform encephalopathies: transmissible cerebral amyloidoses caused by unconventional agents Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microghal cells Expression of microtubule-associated protein 2 by reactive astrocytes Laminin and heparan sulphate proteoglycan in the lesioned adult mammalian central nervous system and their possible relationship to axonal sprouting Localization of the CD44 glycoprotein to fibrous astrocytes in normal white matter and to reactive astrocytes in active lesions in multiple sclerosis Ameboid microglia as effecters of inflammation in the central nervous system A role for IGF-I in the rescue of CNS neurons following hypoxic-ischemic injury Changes in ghal cell markers in recent and old demyelinated lesions in central pontine myelinolysis 11984) Dibutvrvl cvclic AMP causes intermediate filament accumulation and actin reorganisation in astrocytes Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and ~rnb~a-fornix transection Brain interleukin 1 and S-10 immunoreactivity are elevated in Down syndrome and Alzheimer disease Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment Reactive oxygen species and the central nervous system (1992) Neuroactive kynurenines in Lyme borreliosis Substance P and astrocytes: Stimulation of the cyclooxygenase pathway of arachidonic acid metabolism Phorbol diester TPA elicits prostaglandin E release from cultured rat astrocytes Leukotriene production by cultured astroglial cells Primary rat astroglial cultures can generate leukotriene B4 Recombinant interleukin-I beta stimulates eicosanoid production in rat primary culture astrocytes Neuronal regulation of astroglial morphology and proliferation in vitro Astrocytes: auxiliary cells for immune responses in the central nervous system? Role of astrocytes in compartmentation of amino acids and energy metabolism Brain macrophages synthesize interleukin-1 and interleukin-I mRNAs in vitro Quinolinic acid in cerebrospinal fluid and serum in HIV-I infection: relationship to clinical and neurological status Sustained increases in cerebrospinal fluid quinohnic acid concentrations in rhesus macaques (Macaca muluttu) naturally infected with simian retrovirus type-D Increased cerebrospinal fluid quinolinic acid, kynurenic acid, and L-kynurenine in acute septicemia Cerebrospinal fluid and serum neopterin and biopterin in D-retrovirus-infected rhesus macaques (Mucnca muiutta): relationship to clinical and viral status Increased ration of quinolinic acid to kynurenic acid in cerebrospinal fluid of D retrovirus-infected rhesus macaques: relationship to clinical and viral status Cerebrospinal fluid quinolinic acid concentrations are increased in acquired immune deficiency syndrome Graft-vs.-host disease elicits expression of class I and class II histocompatibility antigens and the presence of scattered T lymphocytes in rat central nervous system Perivascular microglial cells of the CNS are bone marrow derived and present antigen in vivo Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat Infection of human T-lymphotropic virw type I to astrocytes in vitro with induction of the class II major histocompatibility complex Expression of Ia antigen by cultured astrocytes treated with gamma-interferon ) c-fos proto-oncogene expression in astrocytes associated with differentiation or proliferation but not depolarization The nroloneed oresence of elia-derived nexin, an endogenous protease inhibitor, in the hippocampus after ischemia-induced delayed neur&al death Tumor necrosis factor identified in multiple sclerosis brain Immunoregulatory molecules and IL-2 receptors identified in multiple sclerosis brain GFAP mRNA levels following stab wounds in rat brain Biochemical and immunocvtochemical changes in alial fibrillarv acidic protein after stab wounds Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation Immunocytochemical localization of GD3 ganglioside to astrocytes in murine cerebellar mutants Temporal expression of mouse glial fibrillary acidic protein mRNA studied by a rapid in situ hybridization procedure Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus Laminin is induced in astrocytes of adult brain by injury Transforming growth factor-beta 1 in the rat brain: increase after injury and inhibition of astrocyte proliferation Transforming growth factor-beta 1 stimulates expression of nerve growth factor in the rat Reactive gliosis Models of neuronal injury in AIDS: another role for the NMDA receptor? Flavivirus infection up-regulates the expression of class I and class II major histocompatibility antigens on and enhances T cell recognition of astrocytes in vitro Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury Enhanced expression of transforming growth factor 81 in the rat brain after a localised cerebral injury NGF gene expression in actively growing brain glia Novel astrocytic protein in multiple sclerosis plaques Production of hemopoietic colony-stimulating factors by astrocytes Permissive and non-permissive reactive astrocytes: immunofluorescence study with antibodies to the glial hyaluronate-binding protein Astroglial cell contributions to neuronal survival and neuritic growth Synthesis and release of neuroactive substances by glial cells Viral particles induce Ia antigen expression on astrocytes Tumor necrosis factor amplifies measles virus-mediated Ia induction on astrocytes Analysis of Ia induction on Lewis rat astrocytes in vitro by virus particles and bacterial adjuvants Hyperinducibility of Ia antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis Immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to Ia-positive cells with dendritic morphology beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity Fibroblast growth factor and glutamate: opposing actions in the generation and degeneration of hippocampal neuroarchitecture Glia protect hippocampal neurons against excitatory amino acid-induced degeneration: involvement of fibroblast growth factor. ht Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes The regulation of proenkephalin expression in a district population of glial cells Induction of interleukin-1 and tumor necrosis factor alpha in brain cultures by human immunodeficiency virus type 1 A novel type of glial cell associated with nodes of Ranvier in rat optic nerve Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct Spinal Cord Reconstruction Immunohistochemical determination of protein kinase C expression and proliferative activity in human brain tumors Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes Immune response gene products (Ia antigens) on glial and endothelial cells in virus-induced demyelination Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1 Accumulation of extracellular glutamate and neuronal death in astrocyte-poor cortical cultures exposed to glutamine Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture Ia expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured T cell lines in mice Cytokine-induced expression of intercellular adhesion molecule-l (ICAM-1) in cultured human oligodendrocytes and astrocytes Expression and induction of intercellular adhesion molecules (ICAMs) and major histocompatibility complex (MHC) antigens on cultured murine oligodendrocytes and astrocytes Heterogeneous induction of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes Production of tumor necrosis factor-aloha bv microglia and astrocytes in culture Glial fibrillary acidic protein and vimentin in the experimental glial reaction of the rat brain Gomori-positive astrocytes: biological properties and implications for neurologic and neuroendocrine disorders Rat astrocytes express interferon-gamma immunoreactivity in normal optic nerve and after nerve transection Immunohistochemical study of glial reaction and serum-protein extravasation in relation to neuronal damage in rat hippocampus after &hernia Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions Tumor necrosis factor mediates myelin and oligodendrocyte damage in oitro Alterations in plasma lipoproteins and apolipoproteins in experimental allergic encephalomyelitis Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis Neuroscience. Vehicles of inactivation Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures and in uiuo Periventricular Gomori-positive glia in brains of X-irradiated rats Rat ependyma and microglia cells express class II MHC antigens after intervenous infusion of recombinant gamma interferon Bilirubin is an antioxidant of possible physiological importance Macrophages in the peripheral nervous system and astroglia in the central nervous system of rat commonly express apolipoprotein E during development but differ in their response to injury Glial uptake of excitatory amino acids influences neuronal survival in cultures of mouse hippocampus Ia-restricted encephalitogenic T lymphocytes mediating EAE lyse autoantigenpresenting astrocytes Coronavirus infection induces H-2 antigen expression on oligodendrocytes and astrocytes Increase in basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells The Cytokine Handbook Acidic fibroblast growth factor-like immunoreactivity in brain of Alzheimer patients Regulation ofplasminogen activators and type-I pfasminogen activator inhibitor by cyclic AMP and phorbol ester in rat astrocytes Multiple sclerosis: involvement of interferons in lesion pathogenesis 19%) interferon-gamma and Ia antigen are present on astrocytes in active chrontc multiple sclerosis lesions On the presence of &positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation Neurotrans~itter t&porters (plus): a promising new gene family nexin-II. a potent aatichymotrypsin, sbbws identity to'amyloid beta-protein precursor lntrathecal application of interferon gamma. 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