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J D Sedgwick

Publications and source records attributed to J D Sedgwick.

At least 55 records · Page 3Linked to original sources
PubMed1996

Microglia induce CD4 T lymphocyte final effector function and death.

Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.

Animals↗
PubMed1996

Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.

Dispase was used to digest central nervous system (CNS) tissue for isolation of the fixed tissue macrophage population (CNS microglia) and other leucocytes present. An apparent reduction in expression of some leucocyte cell surface markers was investigated further by addition of CD45b allotype-marked post-activated CD4+ T cells of known phenotype to CNS tissue preparations derived from a CD45a-expressing rat strain, before enzymatic treatment. Assessment of these T cells for expression of a range of surface molecules after completion of the isolation procedure revealed almost complete loss of expression of CD4 and CD25 and reduced expression of a number of other surface antigens.

Animals↗
PubMed1996

Tolerance to rat liver allografts. III. Donor cell migration and tolerance-associated cytokine production in peripheral lymphoid tissues.

The aim of this work was to investigate the mechanism of spontaneous rat liver allograft tolerance. Liver allografts from a LEW donor into DA recipient (LEW-->DA) or of PVG-->DA were spontaneously tolerated (TOL) across a complete MHC mismatch. In contrast, DA-->LEW or PVG-->LEW liver allografts were rejected in 10 to 15 days (REJ). We examined whether donor cell migration to recipient lymphoid tissues might be associated with TOL. Many donor cells were observed in draining (celiac) lymph nodes (LN) and spleen, reaching a peak on day 1 and then decreasing rapidly thereafter. Irradiation of liver donors, which we have previously shown to delete tolerance, significantly reduced the number of donor leukocytes in recipient lymphoid tissues. While this suggested an association between donor cell migration and tolerance, the number, distribution, and type of donor cells in recipient lymphoid tissues of REJ was similar to those of TOL. Expression of cytokine mRNA in LN and spleen showed an early increase in the expression of IL-2 and IFN-gamma mRNA on day 1 and then a rapid decrease to constitutive levels. Spleen and LN levels of IL-6, IL-10, TNF-alpha, or TGF-beta mRNA showed much less up-regulation than IL-2 or IFN-gamma. Paradoxically, there was greater expression of IL-2 and IFN-gamma mRNA in TOL lymphoid tissues than in REJ, and this superinduction was partially prevented by donor irradiation. Superinduction of IL-2 and IFN-gamma was, therefore, more closely associated with TOL than was donor cell migration. This was confirmed by treatment of TOL recipients with a short course of methylprednisolone, which reduced survival of subsequent donor strain skin grafts. This finding has implications for treatment of human liver transplants and is evidence for a novel pathway of transplant tolerance.

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PubMed1996

Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina.

Recent studies demonstrated that administration of a p55-tumor necrosis factor (TNF) receptor IgG-fusion protein (TNFR-IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen-specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR-IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR-IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR-IgG-treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluorescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ alpha beta TCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.

Animals↗
PubMed1996

Tumour necrosis factor and lymphotoxin: molecular aspects and role in tissue-specific autoimmunity.

Tumour necrosis factor (TNF) is a highly potent, proinflammatory cytokine with broad-ranging functions from the regulation of endothelial cell adhesion molecules to facilitate entry of leucocytes into tissues, to direct induction of cellular cytotoxicity. This diversity of function potentially attributable to TNF in the genesis of inflammatory disorders place TNF as a primary candidate for clinical targeting and considerable success in this regard has been achieved, particularly in rheumatoid arthritis (RA). In this article we provide a short overview of TNF and its homologue lymphotoxin (LT) alpha and beta. Particular emphasis is placed on recent discoveries regarding the cell surface expression of these cytokines and the role of TNF/LT in experimental autoimmune encephalomyelitis (EAE), an animal model of the human demyelinating disease, multiple sclerosis (MS).

Animals↗
PubMed1995

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Animals↗
PubMed1995

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.

Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells. CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2.

Animals↗
PubMed1995

Gene knock-out technology: a methodological overview for the interested novice.

Gene targeting by homologous recombination is a powerful technique, generating mouse strains with defined mutations in their genome. These genetically modified, 'designer' animals allow us for the first time to ask simple questions about elaborate and complex biological systems. Dissecting the function of individual components of the immune system is a perfect application of this technology. Although the techniques involved in the generation of gene knock-out mice are increasingly well defined, to many immunologists the language and concepts are confusing. This review presents the essentials of the technology in a form digestible by the non-expert.

Animals↗
PubMed1995

Immune surveillance and autoantigen recognition in the central nervous system.

The central nervous system (CNS) is considered to be a severely disadvantaged site for the elicitation of immune responses. First, there is no specialised lymphatic drainage. Second, both glia and neuronal cells normally do not express appreciable levels of major histocompatibility complex molecules. Third, the vasculature of the CNS is, at least in most places, lined by endothelial cells that have tight junctions and form a barrier (the blood-brain barrier) against most molecules and cells present in the circulation. Fourth, the cells most important in the initiation of immune response, the leucocyte dendritic cell, are not present. Nevertheless, the existence of inflammatory diseases of this tissue, occurring naturally as in multiple sclerosis or in animals after peripheral immunisation with CNS autoantigens, indicates that the immune system can access and recognise antigens in this site. How this is achieved has become clearer in recent years and primarily seems to involve extravasation of activated but not resting T cells across the blood-brain barrier, and recognition of antigen on macrophage-like perivascular cells, rather than cells within the CNS parenchyme such as astrocytes or microglia. The processes involved in immunological patrolling of the CNS and development of autoimmune inflammatory disease are reviewed.

Animals↗
PubMed1995

Flow cytometric identification of a minority population of MHC class II positive cells in the normal rat retina distinct from CD45lowCD11b/c+CD4low parenchymal microglia.

AIMS: This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry. METHODS: Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia. RESULTS: Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+. CONCLUSIONS: Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.

Animals↗
PubMed1994

Origin and steady-state turnover of class II MHC-bearing dendritic cells in the epithelium of the conducting airways.

Recent studies have identified a contiguous network of class II MHC-bearing dendritic cells (DC) in the airway epithelium of several species, including humans. This network seems comparable to the epidermal Langerhans cell population, comprising up to 700 DC per mm2 of airway epithelium. Moreover, it accounts for virtually all local immunostaining for class II MHC, suggesting an important role in surveillance for inhaled Ag. This study examines the turnover of these airway DC using a radiation chimera model that uses congenic rats expressing different allotypic variants of CD45, detectable via mAbs. Steady-state bone marrow renewal of the airway DC population (which is continuously depleted by migration of mature cells to draining lymph nodes) was interrupted via x-irradiation or high-dose dexamethasone, after which the resident population declined by 85% over the ensuing 72 h. After transplantation with congenic bone marrow and an initial lag period for graft establishment, the airway DC population was rapidly restored to preirradiation levels. These findings indicate a half-life of < or = 2 days for airway epithelial DC. In contrast, epidermal Langerhans cell half-life was > or = 15 days. The only comparable (short) half-life previously reported for a peripheral tissue DC population, is that derived from the gut wall. This indicates that rapidly turning over DC populations are a unique feature of the major "mucosal" organ systems, which is consistent with these DC playing an important role in surveillance of mucosal tissues for incoming Ag.

Animals↗
PubMed1993

Resident macrophages (ramified microglia) of the adult brown Norway rat central nervous system are constitutively major histocompatibility complex class II positive.

A flow cytometric phenotype for isolated adult central nervous system (CNS) ramified microglia was previously defined (CD45low CD11b/c+) in the Lewis strain rat, that clearly distinguished these cells from all blood-derived leucocytes, the latter being CD45high. Consistent with the reported lack of major histocompatibility complex (MHC) expression in the CNS, isolated microglia were mostly MHC class II-. Employing these phenotypic criteria, we now show that a proportion of microglia in Brown Norway (BN) strain rats are constitutively MHC class II+. In spinal cord, up to 25% of microglia are distinctly positive and most have some level of expression. In situ staining of MHC class II+ microglial cells in BN rats indicates that positive cells are typical of ramified microglia on the grounds of both morphological appearance and anatomical location. In Lewis (LEW) rats, the few MHC class II-expressing cells isolated from the normal CNS are CD45high blood-derived cells and not resident microglia. After infection of both LEW and BN rats with a neurotropic murine hepatitis virus (MHV-JHM), MHC class II was rapidly upregulated on microglia in the BN but not in the LEW strain. In the latter, inflammatory cells were the predominant MHC class II-expressing population. Nevertheless, most microglia in the LEW strain could, after some delay, be induced to express MHC class II after transfer of an experimental autoimmune encephalomyelitis (EAE)-inducing encephalitogenic T cell line. Paradoxically, strains resistant to EAE (exemplified by the BN) contained more constitutive MHC class II-expressing microglia than susceptible ones, when a variety of strains were examined. The results clearly establish that the normal CNS may contain MHC class II-expressing cells that are a resident rather than a transient blood-derived population. It is significant that this expression is strain related, but there is no evidence that microglial cell constitutive MHC class II expression predisposes to EAE susceptibility.

Animals↗
PubMed1993

Mechanism of airway inflammation in asthma.

Airway inflammation has emerged as an important contributor to mechanisms of asthma. Furthermore, the presence of airway inflammation is present even in the absence of severe symptoms. To study the mechanisms by which bronchial inflammation can occur in asthma, a number of models have been developed including the airway response to antigen in allergic subjects. The pattern that has emerged from such studies indicates prompt pulmonary mast-cell activation and the apparent initiation of an inflammatory response. This inflammatory response develops over hours and is important in the later and more persistent development of bronchial obstruction. The eosinophil is an important cell in this process as are proinflammatory cytokines generated from activated lung mononuclear cells. The consequence of this multiple cell, multiple proinflammatory product interaction is the establishment of a self-perpetuating, redundant process by which asthma severity increases.

Asthma↗
PubMed1991

Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.

In addition to the major population of infiltrating leukocytes recovered from inflamed rat central nervous system (CNS), all of which expressed high levels of leukocyte common antigen CD45, many cells were coisolated that were MRC OX42+ (complement receptor 3/CD11b) but expressed low-to-moderate levels of CD45 and major histocompatibility complex (MHC) class I molecules. Most cells from normal CNS, in contrast, lay within this latter, CD45low population. From previous in situ immunohistochemical studies, the fortuitously isolated CD45low cells were probably resident (ramified) microglia. Using irradiation chimeras, we show that resident microglia respond to inflammation by upregulating CD45, CD4, and MHC class I molecules with a minority of these cells increasing their expression of MHC class II molecules. A 3- to 4-fold increase in the number of microglia isolated from inflamed CNS provided indirect evidence that the cells had proliferated. In normal CNS, a very small population of blood-derived CD45high-expressing cells are present; most MHC class II expression is associated with these few cells and not with the resident microglia.

Animals↗
PubMed1991

Major histocompatibility complex-expressing nonhematopoietic astroglial cells prime only CD8+ T lymphocytes: astroglial cells as perpetuators but not initiators of CD4+ T cell responses in the central nervous system.

The potential of cells within the central nervous system (CNS) to initiate T lymphocyte responses is not known and was the subject of this study. Using the ability of virgin T lymphocytes to proliferate in a primary response to allogeneic determinants on antigen-presenting cells (APC), we have examined the capacity of major histocompatibility complex (MHC)-expressing astroglial cells to act as stimulators of primary and secondary T cell responses. Neither freshly isolated astrocytes nor primary astrocyte cultures pretreated with interferon gamma (IFN-gamma) to upregulate MHC class I and II expression stimulated unfractionated lymph node (LN) cell populations in the primary mixed lymphocyte reaction. In mixing experiments, astrocytes did not inhibit the T cell response to allogeneic LN stimulators. Purified responder CD4+ T cells also were not stimulated to proliferate or secrete interleukin 2 (IL-2) by MHC class I- and II-expressing astrocytes. In contrast to their inability to stimulate virgin, alloreactive CD4+ T cells, astrocytes were able to specifically stimulate an alloreactive CD4+ T cell line. Unprimed CD8+ T cells, however, exhibited some weak autonomous proliferation to astrocyte stimulators but this response was only substantial in the presence of exogenous IL-2, the latter predominantly being a CD4+ T cell product. Those CD8+ T cells responding in the presence of IL-2 were mainly T cell receptor alpha/beta+ IL-2 receptor (alpha chain)+, and a majority had shifted from high to low CD45R expression. Given the virtual dependence of CD8+ T cells in these studies, on CD4+ T cell help, and the complete absence of activation of this latter subset by astrocytes, it is clear that in the context of this resident CNS cell, further activation of either T cell subset by astrocytes within the CNS can only follow priming by another type of APC. The implications of these results for the induction of T cell responses in the CNS are discussed.

Animals↗
PubMed1990

Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.

Animals↗
PubMed1989

Isolation of encephalitogenic CD4+ T cell clones in the rat. Cloning methodology and interferon-gamma secretion.

In this report we detail a procedure for the cloning of a rat encephalitogenic T cell line and show that the methods normally employed for other species may not always be applicable. The two important differences to be described are, (i) that in these experiments where the parent T cell lines were generated with thymocytes as presenting cells, splenocytes were not suitable as a source of antigen-presenting or stimulator cells and (ii) semipurified forms of IL-2, specifically that derived from EL4 lymphoma cells, resulted in a much reduced cloning frequency and rate of T cell growth compared with cruder mixtures such as that derived from mitogen-stimulated splenocytes. Functional studies with clones derived from a strongly encephalitogenic (experimental autoimmune encephalomyelitis (EAE)-inducing) T cell line revealed that the clones had a reduced capacity to mediate EAE in recipient rats but were otherwise comparable to the parent line in terms of surface phenotype and fine antigen specificity. In an attempt to begin to identify the type of CD4+ T cells that may induce EAE we tested the clones and lines for secreted interferon-gamma by a sensitive ELISA, and showed that all clones secreted high levels of this factor.

Animals↗