Interleukin-6 production by astrocytes: Induction by the neurotransmitter norepinephrine

Astrocytes contribute to the immunocompetence of the central nervous system (CNS) via their expression of class II major histocompatibility complex (MHC) antigens and the production of inflammatory cytokines such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Of these cytokines, IL-6 is of particular interest because one of its many immune and inflammatory actions is the promotion of immunoglobulin synthesis, and it is thought that IL-6 expression within the brain exacerbates autoimmune diseases of the CNS, which are marked by local immunoglobulin production. Several stimuli induce astrocyte IL-6 expression, including such inducible endogenous factors as IL-1β and TNF-α. We have investigated the possibility that a constitutively present endogenous factor, the neurotransmitter norepinephrine (NE), can induce astrocyte IL-6 production. We report that NE induces both IL-6 mRNA and protein in primary neonatal rat astrocytes, with optimal induction at 10 μM. IL-6 protein induction by NE is comparable to that seen with IL-1β or TNF-α, and NE synergizes with these cytokines for a ten-fold enhanced effect. In contrast to astrocytes, microglia are relatively unresponsive to NE, IL-1β and TNF-α for IL-6 production. Experiments with the β-adrenergic receptor agonist isoproterenol, and α and β-adrenergic receptor antagonists (propranolol, phentolamine, atenolol, and yohimbine) indicate that β₂ and α₁-adrenergic receptors are involved in NE induction of astrocyte IL-6 expression. These results help to further the understanding of neuron-glial interactions, and the role of astrocytes and adrenergic activity in immune responses within the CNS.     

Failure of memory (CD44 high) CD4 T cells to recognize their target antigen in retina

Activated T cells recognize Ag in the retina, an immune privileged tissue, and may mediate autoimmune disease. In contrast, this report asks if resting, Ag-specific CD4⁺ CD44⁺ T cells can recognize Ag expressed in the retina. As a probe for Ag, 3E9 T cells specific for an immunodominant epitope of β-galactosidase (β-gal) were transferred to transgenic (Tg) mice expressing β-gal in retinal photoreceptor cells, or to ROSA26 mice which express β-gal widely. The survival, phenotype, and responsiveness of transferred 3E9 T cells were unaffected by the presence of retinal β-gal, but altered by recognition of β-gal in the ROSA26 mice. Inoculation or induction of activated T cells with specificity for this epitope produced autoimmune uveoretinitis, showing that the retinal β-gal is expressed at immunologically significant levels. We conclude that sequestration provides a substantial barrier to recognition of Ag in quiet retina, and that insufficient Ag leaves the retina for detectable immune recognition outside of the retina.     

Nonactivated astrocytes downregulate T cell receptor expression and reduce antigen-specific proliferation and cytokine production of myelin basic protein (MBP)-reactive T cells

Astrocytes express variable levels of MHC class II antigens depending on their activation status or exposure to certain cytokines, notably IFN-γ. When they are induced to express higher surface densities of MHC class II molecules, astrocytes are capable of stimulating syngeneic myelin basic protein (MBP)-reactive T cells to proliferate at a modest rate and to secrete proinflammatory cytokines, such as TNF-α, in response to antigen. In the present investigation evidence is presented that uninduced astrocytes, whether fresh or established as clones, on which surface MHC class II molecules are expressed at a very low density, promote an antigen-dependent reduction of TCR on the surface of syngeneic T cells. Accompanying this effect on the TCR is an induction of T cell hyporeactivity and little or no production of proinflammatory cytokines. These observations suggest that the ability of the astrocyte, through varying their surface MHC class II molecules, can control the effect of antigen-induced T cell responses. In their normal state of low MHC II expression astrocytes are expected to induce no or partial, rather than full, activation of autoreactive T cells that enter the CNS, resulting in T cell hyporeactivity. Since astrocytes usually diminish the production of proinflammatory cytokines by T cells that enter the CNS, the status and control of MHC class II expression on astrocytes should be important determinants of the suppression or enhancement of in situ immune responses in the CNS.     

Transgenic neural plate contributes neuronal cells that survive greater than one year when transplanted into the adylt mouse central nervous system

Neural plate cells from the early embryo may have a number of important advantages as donor material for the delivery of foreign genes into the diseased adult central nervous system (CNS). Mesencephalic neural plate from transgenic GT4-2 mice was used as a source of marked donor cells to determine whether transgene-expressing embryonic CNS progenitor cells can be used as donor material for implantation into the adult mouse brain. Transgenic mouse embryos from this line express the Escherichia coli β-galactosidase (β-gal) gene throughout early CNS development. At the early somite stage (Embryonic Day 8.5), mesencephalic neural plate tissue from heterozygous embryos was dissected out and either transferred into culture for characterization or immediately implanted into the striatum or lateral ventricle of adult wild-type CD-1 mice. Explants of neural plate tissue possessed intense β-gal activity and produced extensive outgrowth of neurofilament-positive processes after 6 days in vitro. Many β-gal-positive cells migrated away from the explanted tissue mass. Grafts of transgenic neural plate tissue in the normal adult mouse striatum, sampled 2 weeks to 1 year after implantation, possessed healthy β-gal-positive cells. More detailed analysis of grafts 3 months after implantation indicated that most β-gal-positive cells were also immunoreactive for neurofilament and microtubule-associated proteins, two neuron-specific markers. In addition, extensive neurofilament-positive axonal tangles were evident within the grafts among the β-gal-positive cells. Electron microscopic (EM) findings of implanted tissue stained with Bluo-Gal revealed many β-gal-positive neurons received synaptic contacts from other cells. A few donor-derived astrocytes were also found in the grafts by EM analysis. No obvious signs of immunological rejection, or of significant decrease in graft volume, were observed at any age. Some β-gal-positive cells were observed to he up to 230 μm away from the main graft mass in both striatal and intraventricular implantations. These data suggest that the neural plate can contribute a long-surviving population of neuronal and astrocytic cells when transplanted into the adult CNS.     

Astrocytes as antigen-presenting cells. Part II: Unlike H-2K-dependent cytotoxic T cells, H-2Ia-restricted T cells are only stimulated in the presence of interferon-γ

Various studies strongly suggest that astrocytes are potent immune-regulating cells. They can be activated to release prostaglandin E, interleukin-1- and interleukin-3-like factors. Cocultivation of antigen-specific T cell lines and astrocytes results in induction of Ia on astrocytes and antigen-specific proliferation of T cells. In the current study, astrocytes were found to be incapable of serving as stimulator cells when unprimed T lymphocytes were used as responders in syngeneic or allogeneic lymphocyte reactions. However, when interferon-γ (IFN-γ) was added, astrocytes became Ia positive and potent stimulators in both syngeneic or allogeneic lymphocyte responses. In the presence of IFN-γ, astrocytes presented antigens to Ia-restricted T hybridoma cells; in contrast hapten was presented to K^b-restricted cytotoxic cloned T cells by astrocytes in the absence of IFN-γ. Thus, cultured astrocytes do function directly as accessory cells in class I antigen-dependent T cell activation, whereas Ia induction by IFN-γ is necessary to enable them to present antigen to class II antigen-restricted T cells.     

Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia

Granulocyte-macrophage colony stimulating factor (GM-CSF) modulates various functions of monocytes/ macrophages including antigen-presenting capacity. Recently it was found that astrocytes produce GM-CSF in the central nervous system (CNS) and that GM-CSF can induce proliferation and morphological changes of microglia. Here we show that GM-CSF can down regulate the interferon-γ-mediated induction of major histocompatibility complex (MHC) class II antigens in microglia, but not in astrocytes. GM-CSF pretreatment completely prevents myelin basic protein-specific T cell proliferation induced by microglia but not astrocytes. GM-CSF did not affect the cell surface expression on microglia of either MHC class I or cell adhesion molecules. The inhibition of microglial MHC class II expression and antigen-presenting function is specific for GM-CSF, as treatment with a different CSF (interleukin-3) did not modulate microglial phenotype or functional capacity. These data suggest that GM-CSF might be involved in the regulation of immune responses within the central nervous system.     

Autocrine and paracrine regulation of astrocyte function by transforming growth factor-β

Recent evidence indicates that astrocytes have a wide range of functions, usually attributed to cells of the immune system, which are critical for maintaining a balanced homeostatic environment in the central nervous system (CNS). Moreover, these cells are known to participate in inflammatory events within the CNS by secreting cytokines such as transforming growth factor-β (TGF-β). In this study we have investigated the ability of TGF-β to influence astrocyte functions. TGF-β mRNA is constitutively expressed by astrocytes in vitro, and when cultures are stimulated with exogenous TGF-β1 an increase in the expression of this mRNA can be shown, suggesting both autocrine and paracrine regulation. In in vitro assays, TGF-β1 is chemotactic for astrocytes in a dose-dependent fashion and inhibits astrocyte proliferation. These results indicating signal transduction by TGF-β1-prompted studies to explore receptor-ligand interactions on isolated astrocyte populations. In a receptor binding assay, we demonstrate that astrocytes appear to express three distinct TGF-β receptor subtypes with nearly 10 000 receptors per cell. Thus, TGF-β may play an important role in regulating astrocyte functions pivotal to the evolution of intracerebral immune responses including recruitment and activation of glial cells at local inflammatory sites within the CNS.     

Rat astrocytes express interferon-γ immunoreactivity in normal optic nerve and after nerve transection

Astrocytes are pivotal components of immune reactions in the CNS. We further support this notion by the localization of the lymphokine interferon-γ (IFN-γ), which plays an important role during immune responses, to astrocytes in rat optic nerve (ON). Astrocytes identified by glial fibrillary acidic protein immunoreactivity were IFN-γ positive in normal and transected ON while oligodendrocytes did not express IFN-γ immunoreactivity. These findings indicate that astrocytes can generate important signals which orchestrate immunoinflammatory responses in the brain.     

Induction of cytokines in glial cells surrounding cortical β-amyloid plaques in transgenic Tg2576 mice with Alzheimer pathology

β-Amyloid plaque deposition observed in brains from Alzheimer patients, might function as immune stimulus for glial/macrophages activation, which is supported by observations of activated microglia expressing interleukin (IL)-1β and elevated IL-6 immunoreactivity in close proximity to amyloid plaques. To elucidate the mechanisms involved in β-amyloid-mediated inflammation, transgenic mice (Tg2576) expressing high levels of the Swedish double mutation of human amyloid precursor protein and progressively developing typical β-amyloid plaques in cortical brain regions including gliosis and astrocytosis, were examined for the expression pattern of a number of cytokines.Using ribonuclease protection assay, interleukin (IL)-1α,-β, IL-1 receptor antagonist, IL-6, IL-10, IL-12, IL-18, interferon-γ, and macrophage migration inhibitory factor (MIF) mRNA were not induced in a number of cortical areas of Tg2576 mice regardless of the postnatal ages studied ranging between 2 and 13 months. Using immunocytochemistry for IL-1α,β, IL-6, tumor necrosis factor (TNF)-α, and macrophage chemotactic protein (MCP)-1, only IL-1β was found to be induced in reactive astrocytes surrounding β-amyloid deposits detected in 14-month-old Tg2576 mice. Using non-radioactive in situ hybridization glial fibrillary acidic protein (GFAP) mRNA was detected to be expressed by reactive astrocytes in close proximity to β-amyloid plaques. The local immune response detected around cortical β-amyloid deposits in transgenic Tg2576 mouse brain is seemingly different to that observed in brains from Alzheimer patients but may represent an initial event of chronic neuroinflammation at later stages of the disease.     

Enhanced response to antigen within lymph nodes of SJL/J mice that were protected against experimental allergic encephalomyelitis by T cell vaccination

The effects of T cell vaccination on peripheral immune responsiveness are not yet fully understood. We have induced resistance to rat spinal cord homogenate (RSCH)-induced experimental allergic encephalomyelitis (EAE) in SJL/J mice by vaccination with four T cell lines (RZ8, RZ15, RZ16, and A51) which were reactive to myelin basic protein (MBP) but not to proteolipid protein (PLP). The effect was relatively neuroantigen-specific since vaccination with ovalbumin (OVA)-reactive and alloantigen-specific cells did not prevent EAE induction. Alloantigen-reactive cells reduced the rate of relapse. The number of central nervous system (CNS) infiltrates and mean clinical EAE scores were significantly reduced. This is the first report demonstrating T cell vaccination in the SJL/J mouse, a strain in which PLP is the predominant encephalitogen in RSCH. The vaccinating cells were of the memory/effector (CD44^high, CD45RB^low) surface phenotype. We examined the effect of T cell vaccination on lymph node T cell proliferative responses to MBP, encephalitogenic peptides of PLP and MBP, OVA and anti-CD3. With the exception of polyclonal cytokine responses to anti-CD3, which remained unchanged, vaccination led to a 5–10-fold augmentation in all, including background, responses. By comparison with lymph node cell (LNC) responses from naive mice and mice primed with OVA, it appeared that T cell vaccination restored cellular activation levels which had been depleted in peripheral lymphoid tissues of unvaccinated animals with EAE.     

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell–astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca²⁺ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca²⁺ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca²⁺ increase is specifically triggered by the autoreactive CD4⁺ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca²⁺ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell–astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.     

Astroglial IFITM3 mediates neuronal impairments following neonatal immune challenge in mice

Interferon‐induced transmembrane protein 3 (IFITM3) ?plays a crucial role in the antiviral responses of Type I interferons (IFNs). The role of IFITM3 in the central nervous system (CNS) is, however, largely unknown, despite the fact that its expression is increased in the brains of patients with neurologic and neuropsychiatric diseases. Here, we show the role of IFITM3 in long‐lasting neuronal impairments in mice following polyriboinosinic‐polyribocytidylic acid (polyI:C, a synthetic double‐stranded RNA)‐induced immune challenge during the early stages of development. We found that the induction of IFITM3 expression in the brain of mice treated with polyI:C was observed only in astrocytes. Cultured astrocytes were activated by polyI:C treatment, leading to an increase in the mRNA levels of inflammatory cytokines as well as Ifitm3. When cultured neurons were treated with the conditioned medium of polyI:C‐treated astrocytes (polyI:C‐ACM), neurite development was impaired. These polyI:C‐ACM‐induced neurodevelopmental abnormalities were alleviated by ifitm3^?/? astrocyte‐conditioned medium. Furthermore, decreases of MAP2 expression, spine density, and dendrite complexity in the frontal cortex as well as memory impairment were evident in polyI:C‐treated wild‐type mice, but such neuronal impairments were not observed in ifitm3^?/? mice. We also found that IFITM3 proteins were localized to the early endosomes of astrocytes following polyI:C treatment and reduced endocytic activity. These findings suggest that the induction of IFITM3 expression in astrocytes by the activation of the innate immune system during the early stages of development has non‐cell autonomous effects that affect subsequent neurodevelopment, leading to neuropathological impairments and brain dysfunction, by impairing endocytosis in astrocytes. GLIA 2013     

Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3^EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3⁺ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4⁺ cells depleted of Foxp3⁺ Tregs into RAG1^−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25⁺ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.     

Radiation enhances tumor necrosis factor α production by murine brain cells

Astrocytes and microglial cells cultured from murine brain were stimulated to produce tumor necrosis α (TNF) by exposure to lipopolysaccharide (LPS). TNF α production began within 2 h with maximum production between 4 and 8 h after atimulation. Clinically relevant low (2 Gy), but not high (8 Gy), doses of radiation significantly increased TNF production by astrocytes and microglial cells in response to LPS. The radiation effect was even more marked with multiple 2 Gy doses. TNF is cytotoxic for oligodendrocytes and for certain tumor cells. It increases vascular permeability and enhances immune responses as well as having biological effects. It is conceivable that production of TNF by astrocytes and microglial cells during clinical radiation theraphy might influence the responses of tumor and/or normal CNS tissues.     

Expression of cathepsin G-like and α1-antichymotrypsin-like proteins in reactive astrocytes

The central nervous system (CNS) of many different species responds to diverse neurologic injuries with an activation of astrocytes. Yet, the exact function of this reactive astrocytosis is unknown. In this report, mouse astrocytes were activated in vivo by focal penetrating brain injury. Reactive astrocytes were stained with antibodies raised against the serine protease cathepsin G (cat.G), the serine protease inhibitor α₁-antichymotrypsin (ACT), or the astrocytic marker glial fibrillary acidic protein (GFAP). Reactive astrocytes expressing both cat.G-like and ACT-like antigens were found around cerebral would margins between 18 h and 13 days after neural lesions. The injury-induced immunostaining was unaltered by 900 rads of total body irradiation, suggesting that the astroglial reaction was relatively independent of bone marrow-derived cells. The in vivo immunostaining was complemented with biochemical assays on cultured primary astrocytes. A synthetic peptide was used as a substrate in combination with specific inhibitors to identify a proteolytic activity within astroglial lysates and culture supernatants that closely resembles cat.G. This activity increased substantially upon stimulation of astrocytes with dibutyryl cyclic AMP and was neutralized by antibodies raised against cat.G. In a separate report, it was shown that astrocytes also contain an ACT-like inhibitory activity³⁸. The production of ACT- and cat.G-like antigens and activities by activated astrocytes should allow these cells to participate in a number of important biologic processes. Many of these processes may benefit the CNS by assisting in early wound repair. However, astroglial proteases and their inhibitors could also contribute to the pathogenesis of certain neurologic diseases.     

Identification of an interferon-γ-responsive element of a class II major histocompatibility gene in rat type 1 astrocytes

Interferon-γ(IFN-γ) has been shown to induce class II major histocompatibility complex (MHC) antigens on several cell types. Previous analysis of cell lines including a glioblastoma multiforme line by our laboratory has mapped an IFN-γ-responsive element to the upstream −141 to −109 base pair (bp) region of the DRA promoter. Using deletion mutants, this report shows that this same general region (−135 to −109 bp) is important for IFN-γ induction in two other human glioma lines and more importantly in primary astrocytes. We have confirmed that this regulatory region of the HLA-DRA gene is necessary for IFN-γ inducibility in astrocytes using a substitution mutant. Sequences beyond −135 bp do not appear to have any additional positive or negative elements.     

Intracerebral Mycobacterium bovis bacilli Calmette–Guerin infection-induced immune responses in the CNS

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette–Guerin. Granuloma and IFN-γ⁺CD4⁺ T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-γIL-17 double-positive CD4⁺ T cells is unique to the CNS. The major CNS source of TNF-α is microglia, with modest production by CD4⁺ T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3⁺CD4⁺ T cells and PD-L2⁺ dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection.