Astrocytes In Multiple Sclerosis

The prevailing view of the Astrocytic response to injury is that reactive Astrocytes impede the regenerative process by forming scar tissue.

A long-standing view has been that Cytokines diminish Neuronal survival and regeneration by stimulating the formation of Astrogliotic scar tissue.

As the levels of many Cytokines dramatically increase following CNS insult, and as this increase in Cytokine expression precedes the production of the Glial Scar.

However, there is a wealth of data indicating that Cytokines "activate" Astrocytes, and that Cytokine-stimulated Astrocytes can promote the recovery of CNS function.

Supporting evidence demonstrates that Cytokine-activated Astrocytes:

1. Produce energy substrates and Trophic Factors for
   • Neurons and Oligodendrocytes
2. Act as free radical and excess Glutamate scavengers
3. Actively restore the Blood-Brain Barrier
4. Promote NeoVascularization
5. Restore CNS Ionic Homeostasis
6. Promote ReMyelination
7. Stimulate NeuroGenesis from Neural Stem Cells

Accordingly, a re-assessment of Cytokine-activated Astrocytes is necessary. Here, we review studies that promote the thesis that Cytokines elicit potent NeuroProtective and regenerative responses from Astrocytes.

Astrocytes are the first cells that are encountered by T-Cells invading the Central Nervous System (CNS) by crossing the Blood-Brain Barrier.

We show that primary Astrocytes contribute to the Immune privilege of the CNS by suppressing Th1 and Th2 Cell activation, proliferation and effector function.

Moreover, this Astrocyte-mediated inhibition of Th Effector Cells was effective on already activated, proliferating cells. Transforming Growth Factor-beta (TGF-ß) secreted by Astrocytes or T-Cells was not the major factor in the inhibition.

The inhibition of T-Cell proliferation induced by Astrocytes was mainly mediated by upregulation of CTLA-4 on already activated T-Cells, which occurred both with and without cell-cell contact.

Upregulation of the inhibitory molecule CTLA-4 on AutoReactive Th Cells, as mediated by Astrocytes, thus represents a novel mechanism for securing the Immune privilege of the CNS.

Considerable attention has recently been paid to Astrocyte functions, which are briefly summarized.

A large amount of data is available about AdrenoCeptor expression and function in Astrocytes, some of it dating back to the 1970's and some of it very recent.

This material is reviewed in the present paper. The Brain is innervated by NorAdrenergic fibers extending from Locus Coeruleus in the BrainStem, which in turn is connected to a network of Adrenergic and NorAdrenergic Nuclei in the Medulla and Pons.

Contributing to the control of NorAdrenergic, Serotonergic, Dopaminergic and Cholinergic function, both in the Central Nervous System (CNS) and in the Periphery.

In the CNS Astrocytes constitute a major target for NorAdrenergic innervation, which regulates Morphological plasticity, energy metabolism, membrane transport, Gap Junction permeability and Immunological Responses in these cells.

NorAdrenergic effects on Astrocytes are essential during consolidation of episodic, long-term memory, which is reinforced by beta-Adrenergic activation.

GlycoGenolysis and synthesis of Glutamate and Glutamine from Glucose, both of which are metabolic processes restricted to Astrocytes, occur at several time-specific stages during the consolidation.

Astrocytic abnormalities are almost certainly important in the pathogenesis of Multiple Sclerosis and in all probability contribute essentially to inflammation and malfunction in Alzheimer's Disease and to mood disturbances in Affective Disorders.

NorAdrenergic function in Astrocytes is severely disturbed by chronic exposure to Cocaine, which also changes Astrocyte morphology.

Development of drugs modifying NorAdrenergic Receptor activity and/or down-stream signaling is advocated for treatment of several Neurological/Psychiatric Disorders and for NeuroProtection.

Astrocytic preparations are suggested for study of mechanism(s) of action of AntiDepressant drugs and PathoPhysiology of Mood Disorders.

Chemokines are small secreted proteins that are essential for the recruitment and activation of specific Leukocyte subsets at sites of inflammation and for the development and Homeostasis of Lymphoid and NonLymphoid tissues.

During the past decade, Chemokines and their Receptors have also emerged as key signaling molecules in NeuroInflammatory Processes and in the development and functioning of the Central Nervous System.

Neurons and Glial Cells, including Astrocytes, Oligodendrocytes, and Microglia Cells, have been identified as cellular sources and/or targets of Chemokines produced in the Central Nervous System in physiological and pathological conditions.

In this article, we provide an update of Chemokines and Chemokine Receptors expressed by Glial Cells focusing on their biological functions and implications in Neurological Diseases.

Astrocytic production of Nerve Growth Factor (NGF) is increased during inflammation of the Central Nervous System (CNS).

Here we show that cell-cell interaction between primary murine Astrocytes and Myelin Basic Protein (MBP)-specific T-Cell Receptor (TCR) transgenic Th1 and Th2 Cells significantly increased production of NGF.

This upregulation was found to be dependent on Antigen recognition. Neutralization of Cytokines produced in cocultures did not affect NGF production. This novel finding suggests a NeuroProtective role of Astrocytes during T-Cell-mediated inflammation in the CNS.

Recently, it is suggested that Peripheric and Central Immune activation play primary role in Hyperalgesia and Allodynia.

Non-Neuronal Cells that are Immune cells in the periphery and Glia (Microglia Cells, Astrocyte) within the Brain and Spinal Cord can drive Hyperalgesic and Allodynic states.

Microglia and Astrocytes, activated in response to noxious stimuli in the body tissues, in the Peripheral Nerves and also in the Spinal Cord, produce and release proteins called ProInflammatory Cytokines (PIC).

Release of PIC from activated Glia cause excessive release of Excitatory NeuroTransmitters from Synaptic Terminals of primary afferent Neuron and then Spinal Cord Dorsal Horn Pain transmission Neurons to become so HyperExcitable.

However, in addition to this effect, PIC appears to interfere with the functions of the Hippocampus that are involved in Cognition, Memory and Mood.

So PIC are important mediators of enhanced Pain both in the Periphery and in the Central Nervous System. As a new approach, it is important that this sight indicates alteration of targets in Pain management.

Astrocytes regulate the integrity of the Blood-Brain Barrier and influence inflammatory processes in the Central Nervous System.

The ProInflammatory Chemokine Monocyte ChemoAttractant Protein-1 (MCP-1), which is both released by and stimulates Astrocytes, is thought to play a crucial role in both these activities.

Because Astrocytes have been shown to possess Caveolae, Vesicular structures that participate in IntraCellular Transport and signal transduction events, we reasoned that expression of the major structural protein of these Organelles, Caveolin-1, might feature critically in the cellular responses to MCP-1.

To test this hypothesis, Caveolin-1 level was "knocked down" in human Astrocyte cultures by using a small interfering RNA approach.

This method resulted in efficient (>90% loss) and specific knockdown of Caveolin-1 expression while sparring Glial Fibrillary Acidic Protein as well as several other proteins involved in EndoCytosis.

Astrocytes suffering Caveolin-1 loss showed diminished ability to down-modulate and internalize the MCP-1 Receptor (CCR2) in response to exposure to this Chemokine and also demonstrated significantly reduced capacity to undergo Chemotaxis and Calcium flux when MCP-1-stimulated.

The results highlight a potentially prominent role for Caveolae and/or Caveolin-1 in mediating Astrocyte responses to MCP-1, a feature that might significantly dictate the progression of inflammatory events at the Blood-Brain Barrier.

Astrocytes in active lesions of Multiple Sclerosis (MS) express Major HistoCompatibility (MHC) Class II molecules, and may play an important role in the presentation of Antigen to Myelin-specific T-Cells.

However, it has been postulated that Astrocytes are unable to act as Antigen-Presenting Cells (APCs) because they would lack the B7 CoStimulatory molecules to activate these T-Cells.

By using double labeling ImmunoFluorescence staining, we demonstrate that reactive Astrocytes in chronic active plaques of Multiple Sclerosis express the CoStimulatory Molecules B7-1 and B7-2, and hence have the necessary attributes to act as Antigen-Presenting Cells.

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 Cells and Astrocytes.

The role of the MHC Class II TransActivator (CIITA) in Ag presentation by Astrocytes and susceptibility to Experimental AutoImmune Encephalomyelitis (EAE) was examined using CIITA-deficient mice.

And, newly created transgenic mice that used the Glial Fibrillary Acidic Protein promoter to target CIITA expression in Astrocytes.

CIITA was required for Class II expression on Astrocytes. Like Class II-deficient mice, CIITA-deficient mice were resistant to EAE by Immunization with CNS AutoAntigen.

Although T-Cells from immunized CIITA-deficient, but not Class II-deficient, mice proliferated and secreted Th1 Cytokines.

CIITA-deficient Splenic APC presented Encephalitogenic Peptide to purified wild-type Encephalitogenic CD4⁺ T-Cells, indicating that CIITA-independent mechanisms can be used for Class II-restricted Ag presentation in Lymphoid Tissue.

CIITA-deficient mice were also resistant to EAE by adoptive transfer of Encephalitogenic Class II-restricted CD4⁺ Th1 cells, indicating that CIITA-dependent Class II expression was required for CNS Ag presentation.

Despite constitutive CIITA-driven Class II expression on Astrocytes in vivo, Glial Fibrillary Acidic Protein-CIITA transgenic mice were no more susceptible to EAE than controls.

CIITA-transfected Astrocytes presented Peptide Ag, but in contrast to IFN-γ-activated Astrocytes, they could not process and present native Ag.

CIITA-transfected Astrocytes did not express Cathepsin S without IFN-γ activation, indicating that CIITA does not regulate other elements that may be required for Ag processing by Astrocytes.

Although our results demonstrate that CIITA-directed Class II expression is required for EAE induction, CIITA-directed Class II expression by Astrocytes does not appear to increase EAE susceptibility.

These results do not support the role of Astrocytes as APC for Class II-restricted Ag presentation during the induction phase of EAE.

Apoptosis of T-Lymphocytes is a common pathway to terminate AutoImmune Inflammation in the Brain as shown in Experimental AutoImmune Encephalomyelitis (EAE) and in the AutoImmune inflamed human Brain.

To date it is unclear to what extent different Glial Cells are involved in the removal of Apoptotic Cells.

In an in vitro Phagocytosis assay we compared the Phagocytic capacity of rat Microglia Cells Cells to remove Apoptotic Lymphocytes with that of Astrocytes.

Apoptosis was induced in autologous Thymocytes and Myelin Basic Protein (MBP)-specific T-Cells by MethylPrednisolone (MP) or by irradiation.

Apoptotic Cells were then added to Glial Cells that were untreated or prestimulated with Interferon-gamma (IFN-γ), InterLeukin-4 (IL-4), Transforming Growth Factor-beta (TGF-ß), or Tumor Necrosis Factor-alpha (TNF-).

Supernatants were collected from cell cultures to measure their Cytokine secretion. Surface Antigen expression was analyzed by flow cytometry.

Both cell types significantly increased their Phagocytic activity in response to the addition of Apoptotic Lymphocytes when compared to NonApoptotic Cells (p < 0.0001).

Astrocytes removed only up to one third of the number of Apoptotic Lymphocytes ingested by Microglia Cells Cells (p < 0.0001). Microglia Cells significantly increased their Phagocytosis rate after IFN-γ stimulation and decreased it in response to IL-4.

In contrast, Astrocyte Phagocytosis was almost unresponsive to Cytokine stimulation. After interaction with Apoptotic Cells, Microglia Cells secreted significantly less TNF-.

Astrocytic TNF- production was also decreased but not to a statistically significant extent. MHC-Class II expression after Phagocytosis was increased on Microglia Cells but not on Astrocytes.

Both Microglia Cells and Astrocytes are capable of ingesting Apoptotic Cells, but Microglia Cells are much more efficient Phagocytes.

Their Phagocytic capacity is modulated by the local MicroEnvironment and Microglial Immune Function is downregulated after Phagocytosis.

We suggest that in vivo Astrocytes might be activated as Phagocytes once the limit of Microglial Phagocytic capacity has been reached.

Analysis of the mechanisms underlying CNS Immune surveillance and ImmunoPathology have provided new insights into the IntraCerebral regulation of Immune Responses.

Here, Francesca Aloisi, Francesco Ria and Luciano Adorini review the role of CNS Antigen-Presenting Cells and focus on the control of Th1 and Th2 responses by Microglia Cells and Astrocytes.