Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.     

MHC antigen expression and cellular response in spontaneous and induced rejection of intracerebral neural xenografts in neonatal rats

Fetal mouse retinae transplanted to the mesencephalon of neonatal rats generally survive for prolonged periods of time without immune suppression suggesting that such grafts enjoy a degree of immunological privilege. A small, but consistent percentage of these transplants, however, ultimately undergo spontaneous rejection. In addition, rejection can be induced by (1) systemically sensitizing the host to the donor antigens by placing a mouse skin graft or (2) producing a local degenerative process adjacent to the graft by removing the host eye contralateral to the side of the retinal transplant. To elucidate the immunological events that underly spontaneous and induced rejection in this system, we examined the distribution of lymphocytes, astrocytes, microglia, and cells expressing major histocompatibility complex (MHC) antigens in unrejected grafts, in transplants showing spontaneous rejection, and in grafts undergoing induced rejection. In unrejected grafts, increased astrocytic and microglial staining was seen around the photoreceptor layer of the graft and at the graft-host interface, but no lymphocytes and only occasional cells expressing MHC antigens were detected. In contrast, spontaneously rejecting grafts showed widespread MHC, lymphocytic, astrocytic, and microglial immunoreactivity that extended well beyond the limits of the transplant into the surrounding host brain. Skin graft-induced rejection produced a temporally consistent, comparatively localized enhancement of astrocytic, microglial and MHC immunoreactivity and infiltration of lymphocytes. Four to five days after skin grafting, before neural graft rejection was detectable histologically, MHC immunoreactivity was demonstrated within the transplant coinciding with the presence of small numbers of lymphocytes and an increase in microglial staining. By 8 days, grafts had undergone profound necrosis. Intense astrocytosis, microglial staining, MHC immunoreactivity, and perivascular lymphocytic cuffing were present within the graft and at the graft-host interface. With longer survival times, several of these changes were also detected within the visual pathways, suggesting that the regions to which the graft projected were also involved, though in a delayed fashion. After eye removal, the temporal pattern of rejection was more protracted and considerably less uniform than that seen after skin grafting. At 7 days, prominent microglial, astrocytic, and MHC immunoreactivity was seen in the area of distribution of the host optic axons within the superior colliculus and to a lesser extent around the graft itself, however, no infiltration of lymphocytes was detected. With longer survival times, an increasing percentage of grafts showed signs of overt rejection with perivascular cuffing by lymphocytes; however, even at 21 days, a small number of grafts remained free of lymphocytic infiltration, despite the presence of intense MHC, astrocytic, and microglial staining. We conclude that the different rejection models studied may involve fundamentally different triggers of the host immune system, but that in each case MHC expression may be the precedent step to graft rejection.     

Patterns of immune rejection of mouse neocortex transplanted into neonatal rat brain, and effects of host immunosuppression

We studied the histological and immunological characteristics of graft rejection in the rodent central nervous system (CNS) using embryonic mouse neocortex transplanted into the CNS of neonatal rats. Grafts from animals aged 8-145 days (n = 210) were examined using standard histological techniques for demonstrating cell morphology and fiber projections. Immunohistochemical techniques were used to identify graft projections into the host CNS. The incidence of graft rejection was 18% for animals between 18 and 30 days of age, but increased abruptly to 73% for animals older than 30 days. No graft rejection was seen in animals younger than 18 days. In a smaller group of xenograft recipient rats sacrificed at specific time points before and after one month of age, detailed immunohistochemical studies were performed to correlate the histological appearance of the graft with the level of major histocompatibility complex (MHC) class I and II immunoreactivity, and microglial, astrocytic and lymphocytic staining within the graft and host brain. Evidence of mild rejection as manifested by the appearance of scattered lymphocytes within the graft coincided with the development of Class I and II immunoreactivity within the graft and at the graft-host interface, which was demonstrated in some animals as early as 24 days. At 29 days of age, rejecting grafts showed diffuse MHC expression within the graft and at the graft-host interface; in contrast, unrejected grafts failed to show MHC immunoreactivity. Thirty-four day-old grafts often showed severe rejection with perivascular lymphocytic cuffing within the graft and in host parenchyma remote from the graft associated with increased MHC immunoreactivity within the host brain. In grafts older than 34 days there was frequently a violent rejection reaction with disruption of the cytoarchitecture of the graft and surrounding host tissues, and widespread MHC antigen expression. Immunosuppression with cyclosporin A was effective in avoiding rejection. The high incidence of rejection with neocortical xenografts is in striking contrast to the much lower incidence seen with retinal xenografts. This suggests that there are immunological features unique to neocortex which incite host recognition and rejection.     

BN rats do not reject F344 brain allografts even after systemic sensitization

Embryonic brain tissue allografts under many circumstances survive transplantation into the brain. It is generally believed that such grafts will not survive if the host animal is systemically sensitized, by skin grafting or other means, to major histocompatibility complex (MHC) antigens of the donor animal. We have found that F344 brain grafts survive in BN hosts even when the host is systemically sensitized to F344 tissue. Embryonic cerebral neocortex from F344 donors was transplanted into BN host rats (n = 95). Subsequently, the host rats were systemically sensitized with donor skin (n = 25), brain tissue (n = 41), or spleen cells (n = 6) and compared with a control group of rats consisting of allografts with no sensitization or sham procedures (n = 23). Rejection of the transplants in BN rat hosts was not provoked by any of the sensitization methods tested. Minor immunological responses that did not result in rejection were, however, present in many host animals. We did not observe infiltration of W3/13+ T cells and OX8+ cytotoxic lymphocytes in any of the groups. Nevertheless, substantial infiltrations of OX6+ antigen-presenting cells and W3/25+ helper T cells were present. There was also an extensive enhancement of MHC class I immunoreactivity in parts of the grafted tissue developing within the third ventricle, but not for the same type of graft in the lateral ventricle. This increase of MHC class I expression was not accompanied by infiltration of cytotoxic T cells. Our findings thus suggest that neural graft rejection depends on general genetic susceptibility to immune reactions, particularly experimental allergic encephalomyelitis and not only on disparity between donor and host antigens encoded by the MHC. Moreover, enhancement of MHC class I and class II expression within transplanted tissue does not predict graft rejection.     

Long-term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization.

Previous studies have suggested that the incidence of spontaneous rejection among immunogenetically mismatched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron-rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4-6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non-neuronal graft material composed predominantly of xenogeneic glial cells (post-natal day 3, PD3, CD-1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague-Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2-positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no histological signs of rejection or major histocompatibility complex antigen expression in and around the transplant-derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well-integrated 1-month-old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2-positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 "long-term" animals showed evidence of graft rejection. These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune-mediated process is unlikely to be responsible for the time-dependent elimination of the donor astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Survival of intrastriatal xenografts of ventral mesencephalic dopamine neurons from MHC-deficient mice to adult rats

Previous studies of neural xenografts have used immunosuppressive agents to prevent graft rejection. In the present study we have examined the survival of mouse dopamine neurons lacking either MHC class I or MHC class II molecules transplanted into rat brains and the host immune and inflammatory responses against the xenografts. Survival of neural grafts was immunocytochemically determined at 4 days, 2 weeks, and 6 weeks after transplantation by counting tyrosine hydroxylase (TH)-positive cells in the graft areas. In addition, the host immune and inflammatory responses against neural xenografts were evaluated by semiquantitatively rating MHC class I and class II antigen expression, accumulation of macrophages and activated microglia, and infiltration of CD4- and CD8-positive T-lymphocytes. For the negative controls, the mean number of TH-positive cells in rats that received wild-type mouse tissue progressively decreased at various time periods following transplantation. In contrast, intrastriatal grafting of either MHC class I or MHC class II antigen-depleted neural xenografts resulted in a prolonged survival and were comparable to cyclosporin A-treated rats that had received wild-type mouse tissue. These results indicate that genetically modified donor tissue lacking MHC molecules can be used to prevent neural xenograft rejection.     

The blood-brain barrier protects foreign antigens in the brain from immune attack

To examine the role of the blood-brain barrier (BBB) in maintaining immune privilege in the brain, the BBB in the region of stably integrated mouse neural grafts implanted in neonatal rat brains was transiently disrupted by intracarotid infusion of hypertonic mannitol. This led to graft rejection and to prominent expression of major histocompatibility complex (MHC) antigens on cells adjacent to the graft. Grafts in control animals receiving an intracarotid infusion of isotonic saline showed only rare MHC expression and no increased incidence of rejection. Opening the barrier in the absence of a graft caused neither MHC expression nor cellular infiltration within the brain, suggesting that the effects of the hypertonic infusion were not produced by an indirect injury-mediated effect on the host brain. We conclude that the integrity of the blood-brain barrier is an important factor in the relative immune privilege of nonsyngeneic neural grafts.     

Cellular events associated with peripherally induced rejection of mature neural xenografts placed into neonatal rat brains

Various circumstances have brought about a dispute concerning the immunologically priviledged status of the central nervous system (CNS). Using a transplantation paradigm, we have examined the cellular events associated with an experimentally induced focal assault on the CNS by the immune system. Chunks of embryonic mouse cortex were transplanted into neonatal rat brains and allowed to survive for 4 weeks. The adult rats then received a skin graft of donor origin to induce rejection of the transplanted tissue. Animals were sacrificed at various time points and examined histologically and immunocytochemically. Under these circumstances, the transplant is rejected via a first-set rejection response, and astrocytes of donor origin appear to be the primary target of the host immune system. Expression of class I and class II major histocompatibility antigens is noted to correlate with lymphocytic invasion of the transplant.     

Norepinephrine inhibits gamma-interferon-induced MHC class II (Ia) antigen expression on cultured brain astrocytes

Recent evidence that astrocytes can be induced to express the class II major histocompatability (MHC) antigens suggests that these cells may be involved in the development of intracerebral immune responses. The principal inducer of MHC class II antigen (Ia) expression is a soluble lymphokine, gamma-interferon (gamma-IFN). Normally astrocytes do not express significant levels of Ia antigens despite the fact that agents such as gamma-IFN may be present in the central nervous system (CNS). Here we report that a major neurotransmitter, norepinephrine (NE), inhibits, in a dose-response fashion, the ability of gamma-IFN to induce Ia antigen expression on cultured astrocytes derived from newborn BALB/c mice. This finding may indicate that the brain contains inhibitory modulators that serve to prevent the up-regulation of intracerebral immune responsiveness.     

Immunohistochemical analysis on the rejection of xenogeneic brain grafts

The central nervous system (CNS) of mammals has long been thought of as an immunologically privileged site. However, this concept is now changing because the rejection of histo-incompatible neural grafts has been frequently observed in the CNS. In neural transplantation used as therapy for some human neurodegenerative diseases, it is important to determine which factors are related to brain graft rejection. In this study, we examined immunological reactions in brains that had received isogeneic (rat to rat) and xenogeneic (mouse to rat) neural transplants. In the immunohistochemical analysis, antibodies against T cell receptor αβ (R73), macrophage and microglia (0X42), MHC class II antigens (0X6), CD4 (W3/25), CD8 (0X8), NK cell (3.2.3), B cell (RLN-9D3), T cell receptor (TCR) Vβ8.2 (R78), TCR Vβ8.5 (B73) and TCR Vβl0 (G101) were used. At the early stage of both isogeneic and xenogeneic transplantation, a nonspecific inflammatory reaction characterized by macrophage infiltration was observed along the needle track which was produced by the grafting procedure. From the day 7 stage onwards, the non-specific inflammatory reaction was replaced by the specific immune reactions of T cell infiltration, neovascularization and necrosis of xenogeneic grafts. Marked T cell infiltration was detected in the lesions, whereas NK and B cells were not. Quantitative analysis of T cell subsets revealed that both CD4+ and CD8+ T cells were found in the xenogeneic transplants. Microglia became activated and strongly expressed MHC class II antigens at the time of graft rejection. Isogeneic transplants, in contrast, showed no histological characteristics of rejection, and numerous dopaminergic neurons with several neurites were observed in the grafts. Based on these findings, we concluded that T cells are the principal effectors in the rejection of xenogeneic neural grafts, and that activated microglia may have some role in presenting antigens to the infiltrating T cells during the rejection process.     

Allografts of CNS tissue possess a blood-brain barrier. II. Angiogenesis in solid tissue and cell suspension grafts

Angiogenesis and patency of blood vessels were analyzed qualitatively in solid CNS and peripheral tissue syngeneic, allogeneic, and xenogeneic grafts and in individual cell suspension grafts of astrocytes, fibroblasts, PC12, and three additional tumor cell lines placed intracerebrally in adult host mice. Postgrafting survival times were 1 day through 4 weeks. The patency of graft vessels was determined in sections from immersion-fixed tissues incubated to reveal the endogenous peroxidase activity of host red cells trapped within the lumen of blood vessels. Additionally, horseradish peroxidase (HRP) was administered intravenously to live hosts; HRP labels host brain and graft vessels on the luminal surface and reveals the presence or absence of a blood-brain barrier (BBB) within the grafts. The origins of blood vessels supplying solid tissue xenografts were identified immunohistochemically with primary antibodies against host (athymic AKR mice) and donor (fetal Lewis rats) major histocompatibility complex (MHC) class I. Blood vessels supplying solid CNS grafts at 1-7 days post-transplantation were identified ultrastructurally and possessed interendothelial tight junctional complexes; however, they were not perfused with either host blood or blood-borne HRP prior to 8 days. Graft vessels at 10 days were outlined consistently by peroxidase-positive red cells in immersion-fixed material and labeled with blood-borne HRP. These vessels provided a BBB to the circulating HRP and exhibited interendothelial tight junctions. Evidence of angiogenesis within solid anterior pituitary grafts and the variety of cell suspension grafts was obtained prior to 3 days post-transplantation in immersion-fixed preparations; the vessels, with the notable exception of those supplying astrocyte cell suspensions, failed to present a BBB to blood-borne peroxidase. Endothelia in the solid pituitary allografts and the PC12 cell grafts were highly fenestrated and exhibited open interendothelial junctions; those in the tumor and fibroblast cell grafts, for the most part, appeared nonfenestrated, and many possessed open interendothelial junctional complexes. Immunostaining for host and donor MHC class I revealed that donor blood vessels predominate over host vessels in CNS xenografts and supply pituitary xenografts exclusively; in both preparations, donor vessels were not identified within the host CNS. Because cell suspension grafts were derived from endothelia-free preparations grown in culture, blood vessels supplying these grafts were necessarily of host CNS origin and manifested a morphological transformation from a BBB to a non-BBB endothelium. The data suggest that angiogenesis in solid CNS grafts placed into the adult host CNS, compared to similarly placed solid peripheral tissue/cell suspension grafts, is not rapid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endothelial cell class II major histocompatibility complex molecule expression in stereotactic brain biopsies of patients with acute inflammatory/demyelinating conditions

To determine if central nervous system (CNS) microvessel endothelial cells express class II major histocompatibility complex (MHC) molecules in early demyelinating lesions in humans, cerebral white matter (WM) biopsies from patients with acute inflammatory/demyelinating conditions, including 4 with multiple sclerosis (MS), were immunostained for class II MHC and other antigens. Eight of 9 biopsies showed focal MHC class II-positive endothelial cells; there were none in the CNS of 1 of the MS patients at autopsy. There were more vessels with class II-positive endothelial cells in areas with intact WM and gliosis than in areas with active demyelination or control WM; class II-positive endothelial cells in small venules and capillaries were adjacent to transmigrating and perivascular CD4-positive cells. By immunoelectron microscopy, class II molecules were localized to vesicles in endothelial cell cytoplasm, suggesting the potential for antigen processing. Perivascular cells, parenchymal microglia, mononuclear cells and the perinuclear cytoplasm but not the processes of astrocytes were also class II-positive. These data indicate that in acute CNS inflammatory/demyelinating lesions, endothelial cells focally and apparently transiently express class II MHC molecules. This expression implies potential antigen-specific interactions, immunoregulatory or signalling functions in endothelial cells, or it may render them susceptible to CD4-positive cell-mediated cytotoxicity. Thus, class II-positive endothelial cells may have pivotal immunologic roles in initial stages of T cell responses in human CNS WM, particularly in acute MS lesions.     

Timing and patterns of astrocyte migration from xenogeneic transplants of the cortex and corpus callosum

The timing, pattern, and pathway of astrocyte migration were investigated in vivo by transplantation of CD-1 mouse cerebral cortex (E13-14) or corpus callosum (P2-3) into neonatal rat cortex. A monoclonal antibody specific for a mouse astrocyte surface antigen (M2) was used to identify the location of the grafts and the migrated donor astrocytes. Within the host cortex, astrocytes from cortical grafts began migration at post-transplantation day (PTD) 7. Over the next 4 days, the most distant displaced donor cells were found progressively further away from the grafts, migrating at a rate of about 220 microns/day. After PTD 11, the migration rate for the farthest displaced donor cells slowed to 25 microns/day, and the cells appeared to stop at about PTD 16 at a distance of 1,100 microns from the edge of the graft. Astrocytes had a faster migration speed in the white matter and covered a longer distance (5 mm) than those in the gray matter, extending on occasion into the contralateral hemisphere. The patterns of astrocyte migration differed depending on local cues around the transplant. Donor astrocytes that had been implanted into the host cortex migrated toward the host cortical surface, sometimes in several radial lines. Astrocytes from grafts, especially callosal grafts, placed in the subcortical white matter migrated along the host fiber tracts. Many astrocytes transplanted into the hippocampus formed laminar patterns close to the hippocampal neuronal layers. These results suggest that the direction, pattern, and speed of astrocyte migration are influenced by local substrates in the host brain.     

Neonatal host astrocyte migration into xenogeneic cerebral cortical grafts.

Migration of host astrocytes into grafts was investigated by transplantation of rat cortex (E16) into the cortex or midbrain of neonatal mice. Host astrocytes, visualized by the mouse astrocyte-specific antibody, began to invade the grafted cortex during the first week post-transplantation and sequentially migrated substantial distances throughout the graft. Host cells in the grafts which were undergoing immune rejection became hypertrophic. These results have important implications when assessing interactions between host and graft cells.     

Allogeneic grafts of fetal dopamine neurons: immunological reactions following active and adoptive immunizations

To define the importance of adoptive sensitization and duration of graft residence on transplant alloimmunization, behavioral and histochemical parameters were examined in unilaterally 6-OHDA-lesioned F344 rat hosts which received fetal ventral mesencephalic (VM) grafts from Wistar-Furth (WF) donors. In all animals which showed increased rotations after alloimmunization, increased numbers of T cell receptor (TcR) positive, CD8⁺ lymphocytes were detected in the grafts. In addition, an increased density of class I MHC antigens was seen in the graft and in the adjacent host brain. Lesser numbers of CD4⁺, CD11b⁺, and MHCII⁺ positive elements were also seen. Perivascular cuffing was often found in actively immunized animals. An increase in TcR⁺ and MHC class I⁺ elements was also seen in animals only adoptively immunized. The tyrosine hydroxylase positive graft area was also markedly reduced in actively immunized animals and the extent of reduction correlated with the number of cells used for immunization. These studies indicate that established allografts can evade rejection as long as host lymphocytes are not activated against graft alloantigens. In addition, increasing graft residence time in the host and adoptive immunization render the graft more susceptible to subsequent rejection.     

Immunological reactions induced by intracerebral transplantation: evidence that host microglia but not astroglia are the antigen-presenting cells

The immunological reactions to embryonic cerebellar xenografts (n = 16) and allografts (n = 8) in host rat brain were studied after 2, 4, and 6 weeks of survival and compared to a control group consisting of 10 rats with isografts. Indirect immunofluorescence was performed on fresh frozen brain sections using antibodies against antigen presenting cells (Ia/Ox-6+ cells) and T helper (W3/25+) cells. Massive infiltrations of both cell types were found within xenografts. Ia antigen was present in the walls of small vessels near the transplant as well as in the ventricles on supra- and subependymal cells. In host tissue surrounding the grafts, Ox-6+ immunoreactivity was also observed in a population of cells ranging from an irregular rod-like shape with short branching processes to more rounded cell bodies with retracted processes. The appearance of these cells was characteristic of microglia. These cells were GFAP-negative. These cellular reactions were associated with rejection of the grafts. In contrast, the allografts survived, but nevertheless cells expressing Ox-6+ and to a lesser extent W3/25+ immunoreactivity were found along the injection needle tract and in damaged host tissue surrounding the grafts. No Ox-6+ perivascular infiltrations were seen. Some staining was also found within the allografts, mainly associated with damaged tissue. Ox-6+ ramified cells were also observed. Both Ox-6+ and W3/25+ immunoreactivity decreased with the time of survival. Host and donor GFAP-positive astrocytes did not express Ox-6+ molecules, and therefore probably were not involved in presenting antigen to effector cells. The control isografts also survived very well, but nevertheless Ox-6+ and less widespread W3/25+ cells were present in surrounding injured host tissue. Ox-6+ perivascular infiltration was not found in the host brain of animals with isografts. Ox-6+ and W3/25+ immunoreactivities were present primarily in graft areas that appeared damaged, often closely associated with injured host tissue. These results indicate that the process of implantation of grafts and associated brain injury induces enhanced Ia/Ox-6+ immunoreactivity, primarily on microglia in brain parenchyma surrounding grafts, and suggest that host microglia may substantially contribute to the initiation of immune reactions against intracerebral grafts. Despite this predisposition to an immunological response, only in the case of xenografts did these reactions, with the addition of Ox-6+ perivascular cuffing and cell infiltrations within the grafts, lead ultimately to graft rejection.     

Expression of Major Histocompatibility Complex Antigens and Induction of Human T-Lymphocyte Proliferation by Astrocytes and Macrophages from Porcine Fetal Brain

Porcine fetal brain cells are of potential use as donor cells for transplantation therapies of neurodegenerative diseases in humans. Our aim was to determine the immunestimulatory properties of astrocytes and macrophages from porcine fetal brain in vitro. By flow cytometry, freshly isolated porcine fetal brain cells were nonautofluorescent, while primary cultures of these cells, prepared to favor growth of astrocytes and macrophages/microglia, consisted of both an autofluorescent and a nonautofluorescent cell population. The cultured autofluorescent cells had qualities typical of macrophages: CD18 (beta(2) integrin subunit) expression, high granularity, and phagocytic activity. The cultured nonautofluorescent cells stained positive for the astrocyte marker glial fibrillary acidic protein and CD56 (NCAM isoform). While freshly isolated porcine fetal brain cells expressed very low levels of major histocompatibility complex (MHC) class I and no MHC class II antigens, primary culture of these cells resulted in upregulation of MHC class I antigens on astrocytes and macrophages and MHC class II antigens on a subpopulation of the macrophages. Single-cell suspensions prepared from the primary cultures were flow sorted into astrocyte and macrophage populations on the basis of cell granularity and autofluorescence or on the basis of CD56 expression. Pure suspensions (>98%) of astrocytes induced a low proliferative response in human T lymphocytes, as determined by [(3)H]thymidine incorporation after 4 days of coculture. A suspension of 91% macrophages was a strong inducer of human T-cell proliferation, even stronger than allogeneic mononuclear blood cells. For neural xenotransplantation, our findings suggest that depletion of macrophages from the donor-cell suspensions may enhance graft survival by reducing cell-mediated rejection.     

Activation of microglial and endothelial cells in the rat brain after treatment with interferon-gamma in vivo

Activation of microglial cells occurs during the pathogenesis of various neurologic diseases. However, the mechanisms of activation in vivo are still elusive. We infused adult rats intravenously with interferon-γ and demonstrated microglial cell activation after three days of treatment. We show for the first time that microglial cells proliferate inside the brain in response to a circulating cytokine. Microglial cells were induced to express major histocompatibility (MHC) class I and class II antigens and small amounts of leukocyte function-associated molecule 1 (LFA-1, CD11a). On endothelial cells of the brain, MHC class I and class II antigens and intercellular adhesion molecule 1 (ICAM-1, CD54) were enhanced. GLIA 19:181–189, 1997. © 1997 Wiley-Liss, Inc.     

Tumor necrosis factor induces expression of MHC class I antigens on mouse astrocytes

The effect of tumor necrosis factor (TNF) on expression of major histocompatibility complex (MHC) antigens was examined in mouse glial cells in vitro. TNF induced MHC class I, but not class II, antigen expression on the surface of astrocytes but not on oligodendrocytes. Glial cells do not normally express detectable amounts of MHC antigens. Thus TNF may play a role in the immunopathogenesis of neurologic diseases that involve MHC class I-restricted reactions.     

Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro

In order to study microglial cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a microglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ.