Contribution of astrocyte-derived IL-15 to CD8 T cell effector functions in multiple sclerosis

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.     

Immunocytochemical characterization of a new marker of fibrous and reactive astrocytes

A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin. Received: 2 May 1995 / Accepted: 31 July 1995     

A Culture Model of Reactive Astrocytes: Increased Nerve Growth Factor Synthesis and Reexpression of Cytokine Responsiveness

Abstract: Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1β and interferon-γ, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.     

FINE STRUCTURE OF THE SURFACE OF THE CEREBRAL CORTEX OF HUMAN BRAIN

Evidence is presented for the existence of arborizing cytoplasmic processes extending from the surface of the cerebral cortex of human brain into the surrounding fluid medium. These originate from subpial fibrous astrocytes and contain the usual cytoplasmic organelles of those cells. They are bordered by basement membrane. Their occurrence is localized and variable over the cortical surface. They are more prevalent in pathological human material than in "normal" human brain and somewhat more prevalent in the latter than in normal rat cortex. Some additional information is presented regarding the relationship of leptomeninges to the cortical surface. The pia mater does not invariably adhere inseparably to the subjacent layer of fibrous astrocytes as generally assumed at present, nor does it always form a continuous layer over the surface of the brain in the material under study. Both collagen and cytoplasmic extensions of astrocytes intervene between these layers. These findings imply that glial elements of the cortex have direct access to the cerebrospinal fluid.     

Localization of immunoglobulins G, A and M in glial cells of reactive and neoplastic origin

The localization of immunoglobulins G, A and M in glial cells of neoplastic and reactive origin have been investigated by the use of the PAP (peroxidase-antiperoxidase) method on paraffin embedded tissue previously fixed in calcium formol. It has been found, that some glial cells of astrocyte type showed a very intense staining when oligoclonal antibodies to human immunoglobulins G, A and M specific for gamma, alpha, and mu chains were used. The localization of immunoglobulins was disclosed in astrocytes of various morphology; astrocytes with well developed processes, gemistocyte type cells without or only with short and thick cell processes and in small cells with scanty cytoplasm. The number of cells with immunoglobulins localized is very small. No positive results have been noted if the normal brain tissue is concerned. The specificity of the method is discussed.     

Role of CD23 in astrocytes inflammatory reaction during HIV-1 related encephalitis

Soluble factors released by intra-cerebral activated cells are implicated in neuronal alterations during central nervous system inflammatory diseases. In this study, the role of the CD23 pathway in astrocyte activation and its participation in human immunodeficiency virus-1 (HIV-1)-induced neuropathology were evaluated. In human primary astrocytes, CD23 protein membrane expression was dose-dependently upregulated by gp120. It was also upregulated by gamma-interferon (gamma-IFN) and modulated by interleukin-1-beta (IL-1beta) whereas microglial cells in these stimulation conditions did not express CD23. Cell surface stimulation of CD23 expressed by astrocytes induced production of nitric oxide (NO) and IL-1beta which was inhibited by a specific inducible NO-synthase (iNOS) inhibitor (aminoguanidine), indicating the implication of this receptor in the astrocyte inflammatory reaction. On brain tissues from five out of five patients with HIV-1-related encephalitis, CD23 was expressed by astrocytes and by some microglial cells, whereas it was not detectable on brain tissue from five of five HIV-1-infected patients without central nervous system (CNS) disease or from two of two control subjects. In addition, CD23 antigen was co-localized with iNOS and nitrotyrosine on brain tissue from patients with HIV1-related encephalitis, suggesting that CD23 participates in iNOS activation of astrocytes in vivo. In conclusion, CD23 ligation is an alternative pathway in the induction of inflammatory product synthesis by astrocytes and participates in CNS inflammation.     

Spontaneous reactive astrogliosis in the dentate gyrus of Bax-deficient mice

Astrocytes play critical roles in many aspects of brain functions via modulation of neurotransmission, metabolism, and structural remodeling in response to physiological or pathological stimuli. Activation of astrocytes is a common phenomenon in many brain pathologies such as stroke, trauma, and neurodegenerative diseases. In this study, we found that gene deletion of the pro-apoptotic gene Bax (Bax-knockout) resulted in a spontaneous reactive astrogliosis in the dentate gyrus, as evidenced by the increased number/volume of astrocytes and cytoplasmic localization of the Olig2 protein. On the other hand, there was no evidence for microglial activation in the dentate gyrus of Bax-knockout mice. Previously, we reported that Bax-knockout mice failed to execute programmed cell death of adult-produced neurons, but the surplus neurons eventually impaired normal synaptic connections and dendritic arborization of dentate gyrus neurons. Therefore, we propose that the reactive astrocytes in the Baxknockout mice may play a role in tissue remodeling of the dentate gyrus following a failure in the programmed cell death of adult-produced neurons.     

Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin

In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.     

The Neuron-Astrocyte-Microglia Triad in Normal Brain Ageing and in a Model of Neuroinflammation in the Rat Hippocampus

Ageing is accompanied by a decline in cognitive functions; along with a variety of neurobiological changes. The association between inflammation and ageing is based on complex molecular and cellular changes that we are only just beginning to understand. The hippocampus is one of the structures more closely related to electrophysiological, structural and morphological changes during ageing. In the present study we examined the effect of normal ageing and LPS-induced inflammation on astroglia-neuron interaction in the rat hippocampus of adult, normal aged and LPS-treated adult rats. Astrocytes were smaller, with thicker and shorter branches and less numerous in CA1 Str. radiatum of aged rats in comparison to adult and LPS-treated rats. Astrocyte branches infiltrated apoptotic neurons of aged and LPS-treated rats. Cellular debris, which were more numerous in CA1 of aged and LPS-treated rats, could be found apposed to astrocytes processes and were phagocytated by reactive microglia. Reactive microglia were present in the CA1 Str. Radiatum, often in association with apoptotic cells. Significant differences were found in the fraction of reactive microglia which was 40% of total in adult, 33% in aged and 50% in LPS-treated rats. Fractalkine (CX3CL1) increased significantly in hippocampus homogenates of aged and LPS-treated rats. The number of CA1 neurons decreased in aged rats. In the hippocampus of aged and LPS-treated rats astrocytes and microglia may help clearing apoptotic cellular debris possibly through CX3CL1 signalling. Our results indicate that astrocytes and microglia in the hippocampus of aged and LPS-infused rats possibly participate in the clearance of cellular debris associated with programmed cell death. The actions of astrocytes may represent either protective mechanisms to control inflammatory processes and the spread of further cellular damage to neighboring tissue, or they may contribute to neuronal damage in pathological conditions.     

Activation of astrocytic lysosomal proteinases by factors released by mononuclear leukocytes

Lysosomal proteinases are increased in the tissue lesions of experimental allergic encephalomyelitis and have been implicated in the degradation of myelin proteins. The cellular origins of the increased proteinases are not known but reactive astrocytes found in areas of increased activity are candidate cells. To evaluate the potential of astrocytes as the source of these proteinases, cathepsin B (CB) and cathepsin D (CD) levels were measured in lysates of cultured astrocytes from neonatal rats. Because astrocytes are activated by inflammatory mediators in demyelinating lesions the effect of activation on proteinase levels was examined. Culture supernatants from mononuclear leukocytes stimulated with either concanavalin A or phytohemagglutinin (PHA) induced significant increases in the astrocytic proteinases. Neither PHA alone, interleukin-1, interleukin-2, nor gamma-interferon induced significant increases. Fractions of the supernatant from PHA stimulated mononuclear leukocytes were tested and activity was found in fractions corresponding to a molecular weight of 45–50,000. These studies demonstrate that astrocytes contain significant amounts of CB and CD activity which can be increased by a factor or factors released by activated mononuclear leukocytes.Preliminary results presented at the 18th annual meeting of the Society for Neuroscience, Toronto, Canada, Nov. 14, 1988.     

Expression of thrombospondin-1 and CD36 and CD47 receptors in the rat brain after exposure to damaging factors in the early postnatal period

The expression of thrombospondin-1 (TS-1) and its receptors CD47 and CD36 in the cerebral cortex and hippocampus of rats under damaging factors in the early postnatal period was studied. After hypoxia on the 7th day of postnatal development, an increase in the number of CD47-expressing cerebral endothelial cells (days of postnatal development: P28–P70) and reduction in the number of TS-1-expressing astrocytes in the cortex at P28 were observed. In animals subjected to early postnatal stress at the age of P2–P15, a decrease in TS-1-expressing astrocytes in the cortex and hippocampus was registered (predominantly at the age of P28). It was noted that these changes characterize the period of long-term effects (P28–P70) of early stress that is relevant to the processes of reparative angiogenesis and arresting of neurological deficits.     

Localization of NADPH diaphorase in the central nervous system of the bivalve mollusc Cristaria tuberculata

By histochemical and electron histochemical methods, NADPH-diaphorase was discovered in neurons and their processes of all ganglia of the central nervous system (CNS) of Cristaria tuberculata. Small cells predominated among neurons containing NADPH-diaphorase. Ultrastructural localization of the enzyme was detected on the perinuclear membrane and membranes of endoplasmic reticulum, cytoplasmic granules and cytosol. In the majority of cells, the reaction product was commonly found in cytoplasmic granules-cytosomes. We studied peculiarities of synaptic contacts between nitrogen oxide synthesizing neurons and their processes. In active synaptic areas, a sediment was discovered on the internal surface of pre- and postsynaptic membranes.     

Comparative aspects of structural organization of astrocytes of the layer i of the human and rat brain cortex

Importance of study of astrocytes for fundamental biology and medicine is due to their key role in formation of the brain barrier system. On taking into consideration the controversial data on structure of the mammalian neocortex superficial layers, of great actuality are the comparative studies of the structural and cytochemical organization of astrocytes in human and in the laboratory animals used in the experimental studies connected with modeling of brain diseases and traumas. The goal of the present work was to study structural organization of astrocytes in the human and rat neocortical layer I. The work was carried out on the autopsy and experimental material from Wistar rats. Astrocytes were revealed immunocytochemically by using antibodies to GFAP, vimentin and nestin. The preparations were examined with aid of light and confocal laser microscopy. No significant difference in the sizes of perinuclear areas were established between the rat and human astrocytes. In the majority of cortex regions, the spectrum of intermediate filaments-forming proteins in these cells was identical. However, there were essential differences revealed in organization of the superficial glial limiting membrane (SGLM). The human SGLM is formed by interlacing of thin processes in the layer I processes, whereas the rat SGLM is represented by specialized astrocytes spread along the cortical surface and connected with the wide-blade processes. The human layer I astrocytes have translaminar processes passing via several cortical layers, whereas in rats such processes are located within the limits of one layer. The revealed differences in the astrocyte structural organization should be taken into account when interpreting results of experimental studies carried out on rats and extrapolating these results to human.     

Localization of endothelin-A and -B receptors during the postnatal development of rat cerebellum

Intense expression of mRNA of endothelin-B receptor (ETBR) has been detected in the Bergmann glia of cerebellum by in situ hybridization, but the intracellular localization has not been reported because of the absence of a useful antibody for immunohistochemical investigations. We made polyclonal antibodies against the carboxyl terminus of human ETBR (420-442) and ETAR (403-427), and performed light- and electron-microscopic immunohistochemistry of the wild-type and ETBR-deficient (sl/sl) rat cerebella. Localization of ETBR during postnatal development was examined by double-staining immunofluorescence using antibodies against ETBR and S-100 beta. In the wild-type rats, ETBR immunoreactivity appeared from postnatal day 5 (P5) and was distributed diffusely in the processes and cell bodies of S-100 beta-positive glial cells. By P14, ETBR immunoreactivity was concentrated in the Golgi apparatus of Bergmann glial cell soma and the plasma membrane of its processes. The ETBR-positive astrocytes in the granular layer decreased in number during P7-14 and had disappeared by week 3. At 3 weeks, ETBR immunoreactivity was restricted to the Golgi apparatus of Bergmann glia. In the sl/sl rats, ETBR immunoreactivity was not observed at all. In contrast to ETBR, ETAR immunoreactivity appeared transiently in the cytoplasm of all astrocytes (Bergmann glia and astrocytes in the granular layer) in the 9- to 14-day-old wild rats and 7- to 14-day-old sl/sl rats, and disappeared within 3 weeks in both. Granule cells did not express immunoreactivity for ETBR and ETAR from the neonatal stage to adulthood. Changes in the intracellular localization of ETBR and transient expression of ETAR may be correlated with the changes of glial functions and proliferation during postnatal development of rat cerebellum.     

Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

Comparative aspects of structural organization of astrocytes of the first layer of the human and rat cerebral cortex

Importance of study of astrocytes for fundamental biology and medicine is due their key role in formation of the brain barrier system. On taking into consideration the controversial data on structure of the mammalian neocortex superficial layers, of great actuality are the comparative studies of the structural and cytochemical organization of astrocytes in human and in the laboratory animals used in the experimental studies connected with modeling of brain diseases and traumas. The goal of the present work was to study structural organization of astrocytes in the human and rat neocortical layer I. The work was on the autopsy and experimental material from Wistar rats. Astrocytes were revealed immunocytochemically by using antibodies to GFAP, vimentin and nestin. The preparations were examined with aid of light and confocal laser microscopy. No significant difference in the sizes of perinuclear areas were established between the rat and human astrocytes. In the majority of cortex regions, the specter of proteins forming intermediate filaments in these cells was identical. However, there were essential differences revealed in organization of the superficial glial bordering lamina (SGBL). The human SGBL is formed by interlacing of thin processes in the layer I processes, whereas the rat SGBL is represented by specialized astrocytes spread along the cortical surface and connected with the wide-blade processes. The human layer I astrocytes have translaminar processes passing via several cortical layers, whereas in rats such processes are located within the limits of one layer. The revealed differences in the astrocyte structural organization should be taken into account when interpreting results of experimental studies carried out on rats and extrapolating these results to human.     

ELECTRON MICROSCOPIC OBSERVATIONS OF THE CENTRAL NERVOUS SYSTEM

In order to establish criteria for the identification of the neural and glial cells of the central nervous system, sections of the brains and spinal cords of mice, rabbits, guinea pigs, and rats; and portions of tumors of the human brain have been examined by electron microscopy. Identification of neurons is made possible by the characteristic cytoplasmic picture, in which there is a distinct granular and less constant membranous ergastoplasmic pattern. In no other cell of the central nervous system is such a distinct granular component present in the ergastoplasm. The shape of the neuron in electron microscopic preparations is similar to that seen by light microscopy with several dendrites containing a similar cytoplasm arising from the perikaryon. Synapses are relatively common on the surface of the neuron and its dendrites. Microglial cells are relatively small and dense with few processes, and are arranged as perineuronal and perivascular satellites for the most part. Occasionally phagocytized material is present in their cytoplasm. The oligodendroglial cells are identifiable by their position as perineuronal satellites and in the white matter as cells arranged in rows. They have a uniformly round to ovoid nucleus with a pale cytoplasm, which has a sparse, finely granular component and a few small mitochondria. The processes are few and relatively straight when cut in longitudinal section. The predominant cellular type in an oligodendroglioma was similar, with a pale cytoplasm. The astrocytes are variable in appearance. Their nuclei are moderately large, irregularly ovoid, and the cytoplasm adjacent to the nucleus is finely granular and scant. In the protoplasmic astrocytes the cytoplasm has a complicated infolded arrangement with reduplication of the plasma membrane, numerous processes extending radially from the cell and rebranching. To a certain extent this same folded plasma membrane was noted in the fibrous astrocytes. However, their more distant processes were narrowed, relatively straight, and filled with numerous dense fibrils. The processes of the astrocyte often surrounded axons, and other cellular processes, and surrounded some vessels, while attaching to a part of the wall of another vessel. Proliferating cells in experimentally produced gliosis and in astrocytic neoplasms were similar in structure. The ependymal cells and the epithelium of the choroid plexus have a specialized surface with microvillous projections of the cytoplasm covered by the plasma membrane. Cilia in varying numbers are present in both epithelia.     

Astrocytes,cells involved in neuro-immune interactions in the central nervous system

The astrocyte, the major glial cell in the central nervous system, may influence many aspects of inflammation and immune reactivity within the brain. We have established a model of chronically activated T lymphocytes, interacting with neural cells of diverse origin to study the complex immune regulatory system suspected to lead to neuroinflammatory diseases. We show that human astrocytes became reactive following T cell contact, secreting proinflammatory cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinase (TIMP). The altered MMP/TIMP system was shown to be involved in deleterious effects displayed by activated T cells towards human multipotent neural precursers by controlling their sensitivity to T cell-induced Fas-mediated apoptosis. MMP/TIMP was suspected to stabilize Fas at the cell membrane. In a model of mixed rat glial cells in primary culture (astrocytes, oligodendrocytes), activated T lymphocytes induced the collapse of processes and the death of immature oligodendrocytes. These effects were associated with upregulation of Fas at the cell surface of oligodendrocytes and secretion of MMP and TIMP by astrocytes. By amplifying the expression of inflammatory molecules including the MMP/TIMP system, astrocytes appear to be a crucial relay in the deleterious molecular cascade triggered by activated T lymphocytes. Detection of altered MMP/TIMP in patients suffering from myelopathy associated with retroviral infection (HTLV-1) strongly suggests its involvement in the physiopathological process of the disease.