Cerebral Endothelial Activation and Signal Transduction Mechanisms during Inflammation and Infectious Disease

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  • CommentaryCerebral Endothelial Activation and SignalTransduction Mechanisms during Inflammation andInfectious Disease

    Raj N. KalariaFrom the Cerebrovascular Disease Group, Department of

    Psychiatry and the Institute for Health of the Elderly, University

    of Newcastle-upon-Tyne, United Kingdom

    Induction of cell surface molecules on cerebral endothe-lial cells, microglia, oligodendrocytes, choroidal epithelialcells, and (to a lesser degree) astrocytes has been re-ported in a few chronic inflammatory conditions and in-fectious diseases of the central nervous system (CNS).The elucidation of autoimmune mechanisms in multiplesclerosis and its experimental model, experimental aller-gic encephalomyelitis (EAE), has provided knowledge ofthe pleiotropic actions of cytokines and specific cellularinteractions between the circulation-derived immunecells and brain elements. Although stereotypical immuneinteractions are mimicked in the CNS, they do not appearto measure up to what is encountered within systemicorgans. This is due not only to the specialized cellularconsistency of the CNS but also to its isolation behind theblood-brain barrier. It may not, therefore, be surprisingthat the targets, namely endothelial cells or macrophages(microglia) of the brain, do not fully respond to injury orinfection in the same manner as or with the expectedresilience of those in the periphery. However, inflamma-tory responses resulting from infections or injury of theCNS activate the brain endothelium and other nonneuralcells of the brain to various degrees depending on thetype, titer, or strength and duration of exposure to theagent or insult. The activation of these cells may bemodulated by the action of one or more cytokines andrelies on expression of respective cell surface receptors,though cytokines are known to work via receptor-inde-pendent systems. In an effort to understand the immuno-pathogenesis and find rational treatments several rodentand nonhuman primate models of infectious disease ofthe CNS have been devised. Such efforts have furtherbeen rewarded by the use of several mouse knockoutstrains or models deficient in molecules of interest.

    Infections of the CNS by viruses, bacteria, and proto-zoa reveal the presence of inflammatory nodules andperivascular cuffing around blood vessels by inflamma-

    tory cells including infiltrated lymphocytes, monocytes,and microglia. Astrocytes and oligodendrocytes also in-variably become hypertrophic and exhibit hyperplasia.However, the consequences of these agents on neu-rones remain largely unknown. Protozoal infections of theCNS such as cerebral malaria (Plasmodium falciparum),toxoplasmosis (Toxoplasma gondii), and trypanosomiasis(Trypanosoma brucei and Chagas disease) lead tounique immunopathological features, although they alsohave features in common with those apparent in chronicinflammatory conditions such as multiple sclerosis. Themolecular mechanisms involved in these conditions andhow these might impinge on understanding brain inflam-matory mechanisms are largely unexplored topics. Tox-oplasmosis, an obligate intracellular protozoan, has re-cently attracted considerable attention because it is themost common CNS infection producing a mass lesion inacquired immune deficiency syndrome (AIDS). Ten to 25percent of AIDS patients have toxoplasmosis of the CNSand Toxoplasma abscesses are a late complication of HIVinfection associated with a low CD4 cell count. It is wellknown that during chronic infection in AIDS and mousemodels of toxoplasmosis, parasite-specific T lympho-cytes release high levels of the cytokine interferon- (IFN-), which is required to prevent cyst reactivation andlikely initiates several cascades of inflammatory and im-mune responses.

    Specific Actions of Cytokines on CerebralEndothelium and Microglia

    In vitro and in vivo studies demonstrate that cytokinessuch as IFN- and tumor necrosis factor- (TNF-) canreadily induce a variety of critical cell adhesion moleculesas well as the major histocompatibility complex (MHC)antigens in nonneural cells of the CNS. IFN- and TNF-seem equally important stimulators of endothelial cells,

    Accepted for publication March 19, 1999.

    Address reprint requests to Prof. R. N. Kalaria, CBV Path Group, MRCUnit, Newcastle General Hospital, Westgate Road, Newcastle-upon-TyneNE4 6BE, UK. E-mail: r.n.kalaria@ncl.ac.uk.

    American Journal of Pathology, Vol. 154, No. 5, May 1999

    Copyright American Society for Investigative Pathology


  • microglia and oligodendrocytes but not astrocytes orneurones. Both class I and class II MHC molecules maybe readily induced in brains of rats and mice dependingon the dose and duration of the presence of IFN-.1,2 It isnow well recognized that the induction of these mole-cules or adhesion proteins, such as intracellular cell ad-hesion molecule (ICAM-1), in endothelial cells as well asmicroglia depends also on the strain and species ofanimal. For example, IFN- or TNF- can elicit morerobust responses in mice susceptible to EAE or cerebralmalaria than in disease-resistant mouse strains.3 IFN-induced expression of MHC class II was significantlyhigher in mice susceptible to cerebral malaria than inresistant mice. Similarly, site-specific responses havebeen noted with respect to these cytokines. Inasmuch asthere appear to be certain differences in the induction ofMHC antigens in endothelial cells between cerebral andperipheral vessels, it is known that circulation-derivedmacrophages display a more robust antigen-presentingcapacity function than brain-resident microglia.4 The ac-tivation stage of cells is another variable important toachieving the desired outcome. For example, the anti-gen-presenting capacity of activated (but not unacti-vated) microglia to nave T cells could be increased bytreatment with IFN-.5

    The differential actions and the modulation of re-sponses of the cytokines may be achieved via differentreceptors. Deckert-Schluter and colleagues6 previouslyshowed that expression of inducible nitric oxide synthase(iNOS) was necessary by signaling through the 55-kdTNF receptor-1 (TNFR1) rather than the 75-kd TNFR2. Inmice subjected to encephalitis by Toxoplasma gondii in-fection the expression of iNOS protein and mRNA inmicroglia were reduced, whereas that of IFN-, TNF-, orinterleukin-1 (IL-1), as well as the recruitment of im-mune cells in brain, were unaffected in TNFR1-deficientmice. Whether such preferential signaling occurs in thecerebral endothelium is unknown. In accord with thedifferential effects of TNFR, however, tissue factor syn-thesis was regulated through TNFR1 but not TNFR2. Thisstudy also showed that endothelial secretion of tissuefactor occurs via the synergistic action of TNF and vas-cular endothelial growth factor.7

    In this issue of The American Journal of Pathology,Deckert-Schluter et al8 report on the role of IFN-R- andTNFR-mediated immune reactions in the activation ofcerebral endothelial cells in the same mouse model ofToxoplasma encephalitis. Although a strong up-regula-tion of the cell adhesion molecules vascular cell adhesionmolecule (VCAM) and ICAM-1 and the MHC antigenswas observed in wild-type and TNFR-deficient mice withToxoplasma encephalitis, it was not observed in the IFN-R-deficient mice. Similar lack of up-regulation of ICAM-1and its ligand LFA-1 and of the MHC class I and IImolecules was also noted in microglia of the IFN-R-deficient mice compared to the TNFR-deficient or wild-type mice. These observations suggest that the IFN-Rrather than the TNFR signaling pathway is involved inactivation of the cerebral endothelium and microglia inmice with Toxoplasma encephalitis. Whether such signal-ing is specific to the condition remains to be seen but it is

    not unlikely that there will be differences, albeit smallones, between various inflammatory conditions and in-fectious diseases, including cerebral malaria. However,the study also showed that although maximal induction ofVCAM and ICAM was IFN--dependent, the IFN-R-de-ficient mice showed significant up-regulation of VCAMupon infection. This observation suggests that, in addi-tion to IFN-, other factors such as IL-1 (also producedin IFN-deficient mice) may be involved in the up-regula-tion of these molecules. That IFN--independent factorsare important in other CNS infections is evident in anothermouse encephalitis caused by the lymphocytic chorio-meningitis virus, where normal induction of ICAM-1 andVCAM was observed in IFN--deficient mice.8 Thesefindings suggest that there may be considerable down-stream cross-talk between different signaling pathways inresponse to particular pathogenetic mechanisms.

    Deckert-Schluter and colleagues further demonstratedthat the recruitment of immune cells (CD4, CD8, andmacrophages) was not impaired in IFN--deficient miceand was similar to that in wild-type mice.8 They suggestthat the low level of induction of cell adhesion moleculeswas sufficient in IFN--deficient mice to ensure entry ofleukocytes into CNS. However, recruitment of macro-phages but not leukocytes was impaired in the TNFR-deficient mice. This is comparable to the EAE mousemodel where TNF- deficient mice also had impairedrecruitment of macrophages but not of T cells to thebrain.9 These observations imply that some cytokinesmay play divergent roles in guiding the movement ofinflammatory cells into brain parenchyma that may beexplained by different pathogenic mechanisms of suchdiseases.

    Synergistic Actions of Cytokines

    Recent studies with brain endothelial cultures haveshown that IFN- and TNF- also act in synergy with eachother or with IL-1 but it is unclear yet how they mightinteract and differentially regulate downstream signalingpathways. Tanaka and McCarron10 had previouslyshown that TNF- inhibits induction of Ia antigens byIFN- in cultures of cerebral endothelial cells and thatIL-1 may act synergistically with TNF- to down-regulateor alter immune responses in the endothelial cells. Usingprimary cultures of human brain endothelial cells, Dor-ovini-Zis and colleagues11 have clearly shown that unlikeTNF-, IFN- has little or no effect in inducing cell adhe-sion molecules including ICAM-1, VCAM-1, and E selec-tin. However, IFN- and TNF- in combination maximallyinduced ICAM-1. In contrast, IFN- alone readily inducedMHC class II antigens in the endothelial cells and likelyincreased permeability of the endothelium via receptor-independent mechanisms. Interestingly, even in bovinebrain endothelial cells12 endothelin-1 (and probably othervasoactive substances) was induced by the combinedactions of TNF- and IFN-. Therefore, direct actions ofcytokines that may result in a change in tone or perme-ability of the cerebrovascular endothelium should not beoverlooked.

    1312 KalariaAJP May 1999, Vol. 154, No. 5

  • Activation of iNOS and production of nitric oxide (NO)further illustrate the synergistic actions of cytokines. Thecombination of IFN- and TNF- (with or without IL-1)was required to induce iNOS and its mRNA in brainendothelial cells and in microglia from rats and mice.Such induction occurs via protein tyrosine kinase and thetranscription factor NF-B. IFN-, in concert with TNF-,also markedly enhances the ability of IL-1-primed en-dothelial cells to release reactive nitrogen intermediates,which may affect vascular tone and permeability. Thesynergistic actions of cytokines such as IFN and TNF areundoubtedly important in magnifying their biological ef-fects during brain injury and inflammation. For example,IFN- can induce TNFR1 and TNFR1 mRNA in cerebralendothelial cells from EAE-susceptible mice.13 In supportof this, Deckert-Schluter et al8 also demonstrated thatTNF mRNA was not induced in the IFN--deficient micewith Toxoplasma encephalitis. A recent study suggestingthat induction of TNFR1 by IFN- facilitates the actionsof TNF- at the transcription level shows the importanceof this finding in oligodendrocytes,14 although it is notunlikely that brain endothelial cells may respond in asimilar manner. Agresti et al14 demonstrated that neitherIFN- nor TNF- was capable of inducing MHC class Iregulatory element (MHC-CRE) binding activity when ad-ministered alone. However, following exposure of ratoligo-dendrocytes to IFN-, TNFR1 expression was tran-scriptionally induced by the binding of signal transducerand activator of transcription-1 (STAT-1) homodimers tothe IFN--activated site (GAS) present in the gene pro-moter. The up-regulation of TNFR1 allowed TNF- toinduce binding of NF-B to the MHC-CRE site. Thus,IFN- and TNF- synergistically stimulated interferon reg-ulatory factor (IRF-1) gene expression. IFN- directly in-duced the binding of STAT-1 homodimers to the GASelement, whereas NF-B binding to B sequence wasactivated by TNF- only after IFN- treatment.

    IFN-R and TNFR- Signaling

    Current studies show convincingly that the actions of TNFin endothelial cells are mediated via protein tyrosine ki-nase and protein kinase C (PKC), rather than proteinkinase A (PKA), signaling pathways.15,16 Human micro-vascular endothelial cells exposed to ultraviolet light Birradiation induced ICAM-1 up-regulation and lympho-cyte-endothelium interaction.17 These authors also ob-served that PKC inhibitors rather than PKA inhibitors wereeffective in attenuating this induction and that PKC- wastranslocated from the cytosol to the membrane, indicat-ing enzyme activation. However, IFN- but not TNF-antibodies blocked the irradiation-induced ICAM-1 up-regulation. In another approach, Grau et al3 describedTNF--induced up-regulation of both TNFR1 and TNFR2receptors in brain endothelial cells from cerebral malaria-susceptible but not from resistant mice. PKC inhibitorsblocked the response to TNF- in both mouse strains butan inhibitor of PKA selectively abolished the response toTNF- in the cerebral malaria-resistant mice. Although

    these observations support the importance of TNR-medi-ated PKC-dependent signaling in certain inflammatory con-ditions, they collectively imply divergent signaling pathwaysfor cytokine-induced adhesion molecule expression.

    IFN- and several other cytokines have been found toactivate the Janus kinases (Jaks) and the STAT pro-teins.18 Although similar signaling ought to be present inall cells expected to respond to cytokines, the compo-nents of the signaling pathways have not yet been char-acterized in CNS cells with IFN-R or other cytokinereceptors. Jaks are a unique class of tyrosine kinasesthat associate with cytokine receptors. On ligand binding,they activate (see above) members of the STAT familythrough phosphorylation on a single tyrosine. ActivatedSTATs form dimers, translocate to the nucleus, bind tospecific response elements (eg, GAS) in promotersof target gene, and transcriptionally activate thesegenes. Both positive and negative regulations of the Jak-STAT pathway have been identified. It has also beenshown that STAT-1 knockout mice have impaired IFNsignaling.18

    Convergence of the actions of TNF- and IFN- isthought to occur through the interferon regulatory factor...


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