Sunhee C. Lee

Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-β1 (TGF-β1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-β1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. NGMMA and NG-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.

Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions

Nitric oxide generated by the inducible form of nitric oxide synthase (iNOS) may contribute to the pathogenesis of multiple sclerosis (MS). In this report, we studied postmortem tissues of MS patients for the expression of iNOS by in situ hybridization and immunocytochemistry. Immunocytochemistry for nitrotyrosine, a putative footprint for peroxynitrite formation was also performed. In acute MS lesions, intense reactivity for iNOS mRNA and protein was detected in reactive astrocytes throughout the lesion and in adjacent normal appearing white matter. Staining of macrophages, inflammatory cell infiltrates, and endothelial cells was variable from case to case, but generally detected only in acute lesions. In chronic MS lesions reactive astrocytes at the lesion edge were positive for iNOS whereas the lesion center was nonreactive. Normal appearing white matter demonstrated little reactivity, as did tissues from noninflamed control brains. Staining for nitrotyrosine was also detected in acute but not chronic MS lesions, and displayed a diffuse parenchymal, membranous, and perivascular pattern of immunoreactivity. These results support the conclusion that iNOS is induced in multiple cell types in MS lesions and that astrocyte-derived nitric oxide could be important in orchestrating inflammatory responses in MS, particularly at the blood-brain barrier.

Cytokines: Powerful Regulators of Glial Cell Activation

It is now clear that cytokines function as powerful regulators of glial cell function in the central nervous system (CNS), either inhibiting or promoting their contribution to CNS pathology. Although these interactions are complex, the availability of animals with targeted deletions of these genes and/or their receptors, as well as transgenic mice in which cytokine expression has been targeted to specific cell types, and the availability of purified populations of glia that can be studied in vitro, has provided a wealth of interesting and frequently surprising data relevant to this activity. A particular feature of many of these studies is that it is the nature of the receptor that is expressed, rather than the cytokine itself, that regulates the functional properties of these cytokines. Because cytokine receptors are themselves modulated by cytokines, it becomes evident that the effects of these cytokines may change dramatically depending upon the cytokine milieu present in the immediate environment. An additional exciting aspect of these studies is the previously underappreciated role of these factors in repair to the CNS. In this review, we focus on current information that has helped to define the role of cytokines in regulating glial cell function as it relates to the properties of microglia and astrocytes.

Reactive Nitrogen Intermediates in Human Neuropathology: An Overview

Nitric oxide (NO) is a recently recognized messenger molecule that has been shown to possess pleiotropic properties, including vasodilation, neurotransmission, cytotoxicity and antimicrobial activity. Constitutive and inducible forms of NO synthase (NOS) have been identified. Activation of cNOS releases relatively low levels of NO for short periods of time whereas induction of iNOS releases high levels of NO for extended periods of time. In rodents, iNOS is predominantly found in cells of the monocyte/macrophage series, including microglia, where it is induced by a combination of bacterial products and cytokines. cNOS and iNOS have also been reported in rodent astrocytes. Activation of iNOS in the CNS could be toxic to many different cell types, including neurons and oligodendrocytes. iNOS, however, has been difficult to demonstrate in human peripheral blood cells, suggesting that the regulation of expression of this enzyme in humans is different from that found in rodents. In this overview, we show that in human glial cells cultured in vitro, astrocytes, but not microglia, can be induced by cytokines to express NO-like activity. Bacterial products are without effect, but a combination of IL-1 and TNF alpha or IFN gamma is a potent stimulus. NO production by astrocytes inhibits Cryptococcus neoformans growth in vitro. In vivo, we show in acute multiple sclerosis lesions, intense NADPH-diaphorase activity is present in hypertrophic astrocytes in the lesion center and at the lesion edge, whereas microglia are nonreactive. Increased NADPH-diaphorase activity colocalizes with immunoreactivity for IL-1 and TNF. These results suggests that the induction of reactive nitrogen intermediates in humans differs from that found in rodents, and supports the conclusion that hypertrophic astrocytes are the major source of NO-like activity in the inflamed CNS.

Expression of the translocator protein of 18 kDa by microglia, macrophages and astrocytes based on immunohistochemical localization in abnormal human brain

Aims: Microglia are involved in neurodegeneration, are prime targets for anti‐inflammatory therapy and are potential biomarkers of disease progression. For example, positron emission tomography imaging employing radioligands for the mitochondrial translocator protein of 18 kDa (TSPO, formerly known as the peripheral benzodiazepine receptor) is being scrutinized to detect neuroinflammation in various diseases. TSPO is presumably present in activated microglia, but may be present in other neural cells. Methods: We sought to elucidate the protein expression in normal human central nervous system, several neurological diseases (HIV encephalitis, Alzheimers disease, multiple sclerosis and stroke) and simian immunodeficiency virus encephalitis by performing immunohistochemistry with two anti‐TSPO antibodies. Results: Although the overall parenchymal staining was minimal in normal brain, endothelial and smooth muscle cells, subpial glia, intravascular monocytes and ependymal cells were TSPO‐positive. In disease states, elevated TSPO was present in parenchymal microglia, macrophages and some hypertrophic astrocytes, but the distribution of TSPO varied depending on the disease, disease stage and proximity to the lesion or relation to infection. Staining with the two antibodies correlated well in white matter, but one antibody also stained cortical neurones. Quantitative analysis demonstrated a significant increase in TSPO in the white matter of HIV encephalitis compared with brains without encephalitis. TSPO expression was also increased in simian immunodeficiency virus encephalitis. Conclusions: This report provides the first comprehensive immunohistochemical analysis of the expression of TSPO. The results are useful for informing the usage of positron emission tomography as an imaging modality and have an impact on the potential use of TSPO as an anti‐inflammatory pharmacological target.

IL-1β Regulates Blood-Brain Barrier Permeability via Reactivation of the Hypoxia-Angiogenesis Program

Loss of blood-brain barrier (BBB) integrity is believed to be an early and significant event in lesion pathogenesis in the inflammatory demyelinating disease multiple sclerosis (MS), and understanding mechanisms involved may lead to novel therapeutic avenues for this disorder. Well-differentiated endothelium forms the basis of the BBB, while astrocytes control the balance between barrier stability and permeability via production of factors that restrict or promote vessel plasticity. In this study, we report that the proinflammatory cytokine IL-1β, which is prominently expressed in active MS lesions, causes a shift in the expression of these factors to favor plasticity and permeability. The transcription factor, hypoxia inducible factor-1 (HIF-1), plays a significant role in this switch. Using a microarray-based approach, we found that in human astrocytes, IL-1β induced the expression of genes favoring vessel plasticity, including HIF-1α and its target, vascular endothelial growth factor-A (VEGF-A). Demonstrating relevance to MS, we showed that HIF-1α and VEGF-A were expressed by reactive astrocytes in active MS lesions, while the VEGF receptor VEGFR2/flk-1 localized to endothelium and IL-1 to microglia/macrophages. Suggesting functional significance, we found that expression of IL-1β in the brain induced astrocytic expression of HIF-1α, VEGF-A, and BBB permeability. In addition, we confirmed VEGF-A to be a potent inducer of BBB permeability and angiogenesis, and demonstrated the importance of IL-1β-induced HIF-1α in its regulation. These results suggest that IL-1β contributes to BBB permeability in MS via reactivation of the HIF–VEGF axis. This pathway may represent a potential therapeutic target to restrict lesion formation.

Human Brain Parenchymal Microglia Express CD14 and CD45 and are Productively Infected by HIV-1 in HIV-1 Encephalitis

Microglia are endogenous brain macrophages that show distinct phenotypes such as expression of myeloid antigens, ramified morphology, and presence within the neural parenchyma. They play significant roles in a number of human CNS diseases including AIDS dementia. Together with monocytederived (perivascular) macrophages, microglia represent a major target of HIV‐1 infection. However, a recent report challenged this notion based on findings in SIV encephalitis. This study concluded that perivascular macrophages can be distinguished from parenchymal microglial cells by their expression of CD14 and CD45, and that macrophages, but not microglia, are productively infected in SIV and HIV encephalitis. To address whether parenchymal microglia are productively infected in HIV encephalitis, we analyzed expression of CD14, CD45 and HIV‐1 p24 in human brain. Microglia were identified based on their characteristic ramified morphology and location in the neural parenchyma. We found that parenchymal microglia are CD14+ (activated), CD45+ (resting and activated), and constitute approximately two thirds of the p24+ cells in HIV encephalitis cases. These results demonstrate that microglia are major targets of infection by HIV‐1, and delineate possible differences between HIVE and SIVE. Because productively infected tissue macrophages serve as the major viral reservoir, these findings have important implications for AIDS.

IL-1-regulated responses in astrocytes: relevance to injury and recovery.

In the central nervous system (CNS), the cellular processes of astrocytes make intimate contact with essentially all areas of the brain. They have also been shown to be functionally coupled to neurons, oligodendrocytes, and other astrocytes via both contact‐dependent and non‐contact‐dependent pathways. These observations have led to the suggestion that a major function of astrocytes in the CNS is to maintain the homeostatic environment, thus promoting the proper functioning of the neuronal network. Inflammation in the CNS disrupts this process either transiently or permanently and, as such, is thought to be tightly regulated by both astrocytes and microglia. The remarkable role that single cytokines, such as TNF and IL‐1, may play in this process has now been well accepted, but the extent of the reprogramming of the transcriptional machinery initiated by these factors remains to be fully appreciated. With the advent of microarray technology, a more comprehensive analysis of this process is now available. In this report we review data obtained with this technology to provide an overview of the extent of changes induced in astrocytes by the cytokine IL‐1.

The cytokine IL-1β transiently enhances P2X7 receptor expression and function in human astrocytes

Extracellular nucleotide di‐ and triphosphates such as ATP and ADP mediate their effects through purinergic P2 receptors belonging to either the metabotropic P2Y or the ionotropic P2X receptor family. The P2X7R is a unique member of the P2X family, which forms a pore in response to ligand stimulation, regulating cell permeability, cytokine release, and/or apoptosis. This receptor is also unique in that its affinity for the ligand benzoyl‐benzoyl ATP (BzATP) is at least 10‐fold greater than that of ATP. Primary human fetal astrocytes in culture express low‐levels of P2X7R mRNA and protein, and BzATP induces only a slight influx in intracellular calcium [Ca2+]i, with little demonstrable effect on gene expression or pore formation in these cells. We now show that, following treatment with the proinflammatory cytokine IL‐1β, BzATP induces a robust rise in [Ca2+]i with agonist and antagonist profiles indicative of the P2X7R. IL‐1β also induced the formation of membrane pores as evidenced by the uptake of YO‐PRO‐1 (375 Da). Quantitative real‐time PCR demonstrated transient upregulation of P2X7R mRNA in IL‐1β‐treated cells, while FACS analysis indicated a similar upregulation of P2X7R protein at the cell membrane. In multiple sclerosis lesions, immunoreactivity for the P2X7R was demonstrated on reactive astrocytes in autopsy brain tissues. In turn, P2X7R stimulation increased the production of IL‐1‐induced nitric oxide synthase activity by astrocytes in culture. These studies suggest that signaling via the P2X7R may modulate the astrocytic response to inflammation in the human central nervous system.