Pro-regenerative properties of cytokine-activated astrocytes.

The prevailing view of the astrocytic response to injury is that reactive astrocytes impede the regenerative process by forming 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, a long-standing view has been that cytokines diminish neuronal survival and regeneration by stimulating the formation of astrogliotic scar tissue. 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 produce energy substrates and trophic factors for neurons and oligodendrocytes, act as free radical and excess glutamate scavengers, actively restore the blood-brain barrier, promote neovascularization, restore CNS ionic homeostasis, promote remyelination and also 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.

Interleukin-1 beta is required for the early evolution of reactive astrogliosis following CNS lesion.

The CNS response to injury is characterized by the rapid activation of astrocytes in a process known as astrogliosis. The function of reactive astrocytes is controversial, in that both beneficial and detrimental properties are postulated. Identification of the molecules involved in regulating astrogliosis is an important step towards understanding astrocyte functions and establishing suitable conditions for CNS regeneration. We previously reported that inflammatory cytokines are regulators of astrogliosis but the key cytokine involved in initiating astrogliosis was unclear. We describe here that the elevation of glial fibrillary acid protein (GFAP) transcripts follows the very early rise of interleukin (IL)-1beta mRNA in a murine corticectomy model of CNS lesion. Furthermore, the injury-induced upregulation of GFAP mRNA and protein did not occur in mice genetically deficient for IL-1beta compared to wild-type animals. This was correlated with an absence of an increase in GFAP-immunoreactivity (GFAP-ir) in IL-1beta-null mice at 2 and 3 days of injury. However, by 5 to 7 days after the lesion, GFAP-ir was not different between cytokine-deficient and wild-type controls. Functionally, mice lacking IL-1beta exhibited a significant impairment in reformation of the blood-brain barrier (BBB) following corticectomy compared to wild-type controls. These findings suggest that the rapid production of IL-1beta following trauma plays a beneficial role in initiating astrogliosis in an attempt to restore the integrity of the BBB and seal off the wound site.

P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase.

Leukocyte infiltration in the CNS after trauma or inflammation is triggered in part by upregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), in astrocytes. However the signals that induce the upregulation of MCP-1 in astrocytes are unknown. We have investigated the roles for ATP P2X7 receptor activation because ATP is an intercellular signaling transmitter that is released in both trauma and inflammation and P2X7 receptors are involved in immune system signaling. Astrocytes in primary cell culture and acutely isolated from the hippocampus were immunopositive for P2X7 receptors. In astrocyte cultures, application of the selective P2X7 agonist, benzoyl-benzoyl ATP (Bz-ATP), activated MAP kinases extracellular signal receptor-activated kinase 1 (ERK1), ERK2, and p38. Purinergic antagonists depressed this activation with a profile suggesting P2X7 receptors. Bz-ATP also increased MCP-1 expression in cultured astrocytes, and again P2X7 antagonists prevented this increase. Blocking either the ERK1/ERK2 or the p38 pathway (with PD98059 or SB203580, respectively) significantly inhibited Bz-ATP-induced MCP-1 expression. Coapplication of both antagonists caused a greater depression. We also tested the roles for ATP receptor activation in inducing MCP-1 upregulation in corticectomy, an in vivo model of trauma. This model of cortical trauma was previously shown to increase MCP-1 expression in vivo principally in astrocytes. Suramin, a wide-spectrum purinergic receptor antagonist, significantly depressed the rapid (3 hr) trauma-induced increase in MCP-1 mRNA. These data indicate that purinergic transmitter receptors in astrocytes are important in regulating chemokine synthesis. The regulation of MCP-1 in astrocytes by ATP may be important in mediating communication with hematopoietic inflammatory cells.

Central nervous system-initiated inflammation and neurotrophism in trauma: IL-1 beta is required for the production of ciliary neurotrophic factor.

Injury to the CNS results in the production and accumulation of inflammatory cytokines within this tissue. The origin and role of inflammation within the CNS remains controversial. In this paper we demonstrate that an acute trauma to the mouse brain results in the rapid elevation of IL-1beta. This increase is detectable by 15 min after injury and significantly precedes the influx of leukocytes that occurs hours after. To confirm that IL-1beta up-regulation is initiated by cells within the CNS, in situ hybridization for cytokine transcript was combined with cell type immunohistochemistry. The results reveal parenchymal microglia to be the sole source of IL-1beta at 3 h postinjury. A role for CNS-initiated inflammation was addressed by examining the expression of the neurotrophic factor, ciliary neurotrophic factor (CNTF). Analysis of their temporal relationship suggests the up-regulation of CNTF by IL-1beta, which was confirmed through three lines of evidence. First, the application of IL-1 receptor antagonist into the lesion site attenuated the up-regulation of CNTF. Second, the examination of corticectomized animals genetically deficient for IL-1beta found no CNTF up-regulation. Third, the lack of CNTF elevation in IL-1beta null mice was rescued through exogenous application of IL-1beta into the lesion site. These findings provide the first evidence of the requirement for IL-1beta in the production of CNTF following CNS trauma, and suggest that inflammation can have a beneficial impact on the regenerative capacity of the CNS.

Interleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo.

An acute trauma to the CNS rapidly results in the upregulation of inflammatory cytokines that include interleukin-1 (IL-1). We report here that the levels of several matrix metalloproteinases (MMPs) are also elevated following a corticectomy trauma injury to the mouse CNS. The delayed upregulation of MMPs compared to that for IL-1 suggests the possibility that inflammatory cytokines regulate MMP production in CNS trauma. To resolve this, we developed a method to isolate and maintain highly enriched human fetal neurons or astrocytes in culture and examined the regulation by cytokines of the activity of a subgroup of MMPs, the gelatinases (MMP-2 and -9). While both neuronal and astrocytic cultures displayed comparable MMP-2 activity, as evidenced by gelatin zymography, levels of MMP-9 were proportionately higher in neurons compared to astrocytes. Of a variety of cytokines and growth factors tested in vitro, only IL-1beta was effective in increasing the neuronal expression of MMP-9. Finally, an IL-1 receptor antagonist attenuated the increase of neuronal MMP-9 in culture and abolished the injury-induced increase of MMP-9 in the mouse brain. These results implicate IL-1beta as a key regulator of neuronal MMP-9 in culture and of the elevation of MMP-9 that occurs following mouse CNS trauma.

Mononuclear cell proliferation and hyperplasia during Wallerian degeneration in the visual system of the goldfish in the presence or absence of regenerating optic axons.

Patterns of proliferation and changes in non-neuronal cell number in the visual system of the goldfish have been quantitatively examined during optic axon regeneration after an optic nerve crush (ONC). In addition, in order to examine the effect of the regenerating axons on cellular responses in the visual pathways, we did a similar analysis of animals with the right eye removed (ER). Finally, we used double labeling protocols to demonstrate that the proliferating cells that we were counting were mostly phagocytic cells of the mononuclear lineage. In animals with an ONC, we observed an early burst of proliferation that peaked between 7 and 14 days after surgery in all parts of the visual system. In the optic tract, there was also a secondary rise that peaked at 21 days. Levels of proliferation returned to normal by 32 days postoperative in the tract and tectum, while they remained somewhat elevated in the optic nerve for at least 93 days. The total number of non-neuronal cells in the visual paths also rose to peak values between 7 and 14 days after ONC surgery. In the optic tract and tectum, the values fell rapidly after this time, while in the optic nerve, there was a secondary peak at 32 days after which values remained elevated for the duration of the experiment. As compared to animals with an ONC, enucleation resulted in elevated proliferation and hyperplasia at early postoperative intervals. However, because these differences occurred when axons had not yet regenerated into the affected structures, these data do not provide strong evidence for a direct effect of regenerating optic axons on the early cellular responses during Wallerian degeneration in the goldfish. In addition, in the tectum, there was an early increment in cell number that was not associated with elevated levels of proliferation. We believe that this increment represents immigration of resident microglia from other regions of the brain.

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.