Richard Milner

The integrin family of cell adhesion molecules has multiple functions within the CNS

Integrins comprise a large family of cell adhesion molecules that mediate interactions between the extracellular environment and the cytoplasm. During the last decade, analysis of the expression and function of these molecules has revealed that integrins regulate many aspects of cell behavior including cell death, proliferation, migration, and differentiation. Within the central nervous system (CNS), most of the early studies focused on the role of integrins in mediating adhesive and migratory events in two distinct processes: neural development and CNS inflammation. Interestingly, recent analysis of transgenic mice has provided some surprising results regarding the role of integrins in neural development. Furthermore, a large body of evidence now supports the idea that in addition to these well‐described functions, integrins play multiple roles in the CNS, both during development and in the adult in areas as diverse as synaptogenesis, activation of microglia, and stabilization of the endothelium and blood‐brain barrier. Many excellent reviews have addressed the contribution of integrins in mediating leukocyte extravasation during CNS inflammation. This review will focus on recently emerging evidence of novel and diverse roles of integrins and their ligands in the CNS during development and in the adult, in health and disease.

Microglial Activation and Matrix Protease Generation During Focal Cerebral Ischemia

Local environmental conditions contribute to the activation state of cells. Extracellular matrix glycoproteins participate in cell-cell boundaries within the microvascular and extravascular tissues of the central nervous system and provide a scaffold for the local environment. These conditions are altered during focal cerebral ischemia (and other central nervous system disorders) when extracellular matrix boundaries are degraded or when matrix proteins in the vascular circulation enter the neuropil as the microvascular permeability barrier is degraded. Microglia in the resting state become activated after the onset of ischemia. During activation these cells can express a number of factors and proteases, including latent matrix metalloproteinase-9 (pro–MMP-9). Whereas MMP-9 and MMP-2 are generated early during focal ischemia in select models, their cellular sources in vivo are still under study. In vitro microglia cells activate and respond to exposure to specific matrix proteins (eg, vitronectin, fibronectin) that circulate. Certain MMP inhibitors, specifically tetracycline derivatives, can modulate microglial activation and reduce injury volume in limited studies. But, the injury reduction relies on preinjury exposure to the tetracycline. Other studies underway suggest the hypothesis that microglial cell activation and pro-MMP-9 generation during focal cerebral ischemia is promoted in part by matrix proteins in the circulation that extravasate into the neuropil when the blood-brain barrier is compromised. These matrix proteins are known to activate microglia through their specific cell surface matrix receptors.

The Extracellular Matrix and Cytokines Regulate Microglial Integrin Expression and Activation

Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-α promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the α4β1 and Mac-1 integrins. In contrast, TGF-β1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the α4β1, α5β1 and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-β1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.

Interferon-Independent, Human Immunodeficiency Virus Type 1 gp120-Mediated Induction of CXCL10/IP-10 Gene Expression by Astrocytes In Vivo and In Vitro

ABSTRACT The CXC chemokine gamma interferon (IFN-γ)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3+ T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.

Contrasting effects of mitogenic growth factors on oligodendrocyte precursor cell migration.

We have examined the effects of the mitogenic growth factors platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and glial growth factor‐2 (GGF‐2) on oligodendrocyte precursor migration. In an agarose drop migration assay PDGF and bFGF stimulated migration while GGF‐2 had no effect. The migration‐enhancing effect of bFGF cannot be blocked by neutralising antibodies against PDGF, confirming that this effect is direct and not mediated via upregulation of PDGF receptors. Based on our results, we propose a model in which the differing effects of PDGF and GGF‐2 ensure appropriate numbers of oligodendrocyte precursor cells in the vicinity of axons to be myelinated during development. GLIA 19:85–90, 1997.

Cytokines Regulate Microglial Adhesion to Laminin and Astrocyte Extracellular Matrix via Protein Kinase C-Dependent Activation of the α6β1 Integrin

Microglia are highly plastic cells that participate in inflammatory and injury responses within the CNS and that can migrate extensively after activation. Because astrocytes and their extracellular matrix (ECM) form a large part of the CNS parenchyma, we undertook to study the adhesive interactions between microglia and these substrates in vitro. In contrast to oligodendrocyte precursor cells, microglia formed only weak interactions with astrocytes and their ECM. On specific ECM substrates the microglia adhered strongly to fibronectin, vitronectin, and plastic but only weakly to laminin. Microglial adhesion to laminin was increased significantly by the proinflammatory cytokines TNF, IFN-α, and IFN-γ but was decreased by TGF-β1, with the TGF-β1 effect being dominant over the other cytokines. Fluorescence-activated cell sorting (FACS) analysis and immunoprecipitation showed that microglia constitutively express the α6β1 integrin, a well characterized laminin receptor, and that α6β1 expression levels did not change after cytokine treatment. Function-blocking studies showed that microglial adhesion to laminin is mediated entirely by the α6β1 integrin, strongly suggesting that the cytokine regulation of adhesion to laminin is mediated by changes in the activation state of α6β1. Analysis of signaling pathways revealed that activation of α6β1 is mediated by a PKC-dependent mechanism. In light of the evidence that laminin expression is upregulated after CNS injury, the findings suggest that cytokine regulation of microglial adhesion to laminin may play a fundamental role in determining the extent of microglial infiltration into and retention at the site of injury.

Integrins mediate a neuronal survival signal for oligodendrocytes

Target-dependent survival of newly differentiated cells is an important part of neural development. In the case of myelin-forming oligodendrocytes, it matches the number of oligodendrocytes to the available axons [1]. In addition to growth factors, an axonal signal regulates this survival: when axons are transected, oligodendrocytes die and, conversely, when the number of axons is increased by genetic manipulation, oligodendrocyte numbers increase [2] [3]. Newly formed oligodendrocytes that fail to contact axons undergo apoptosis, and co-culture experiments that model axon-glial interactions in vitro reveal a neuronal survival effect not present in neuron-conditioned medium [4] [5], suggesting that the signal is non-diffusible and present on the surface of axons. The nature of these neuronal signals is unknown, as are the mechanisms by which they interact with growth-factor-mediated survival signals. As integrins can regulate survival in other cell types [6] [7] [8], we determined whether integrins are involved in the neuronal survival effect. We found that the laminin receptor alpha6beta1 integrin, which is expressed on oligodendrocytes, enhances the sensitivity of oligodendrocytes to the survival effect of growth factors. On the basis of this interaction between integrin and growth-factor-mediated signalling, we propose a simple model by which signals from axons and other cell types might interact to regulate oligodendrocyte cell numbers.

Developmental Regulation of β1 Integrins during Angiogenesis in the Central Nervous System

Abstract We examined β1 integrin expression during angiogenesis in the developing mouse CNS. Maturation of blood vessels was accompanied by three main events: (1) marked upregulation of β1 integrin expression; (2) a switch in β1 integrin expression, from α4 and α5 at postnatal day 1 to α1 and α6 in the adult; and (3) downregulation of fibronectin and upregulation of laminin expression. Thus, blood vessel maturation was associated with a developmental switch, from fibronectin-mediated signaling early in angiogenesis, to laminin-mediated signaling at later stages. To investigate the potential role of α4 and α5 integrins in angiogenesis, we developed a novel approach to purify endothelial cells from the CNS by selective adhesion to fibronectin. BrdU incorporation showed that fibronectin induced more endothelial cell proliferation than laminin, and antibody-blocking studies revealed that fibronectin mediated its effect via both α4 and α5 integrins. This work provides the first evidence that there is a developmental switch in the use of β1 integrins during angiogenesis. Furthermore, it suggests that regulation of β1 integrin and ECM expression by endothelial cells are important factors influencing vascular development in the CNS.

Responses of Endothelial Cell and Astrocyte Matrix-Integrin Receptors to Ischemia Mimic Those Observed in the Neurovascular Unit

Background and Purpose— Apposition of endothelial cells and astrocyte foot processes to the basal lamina matrix is postulated to underlie the cerebral microvessel permeability barrier. Focal cerebral ischemia induces rapid loss of select matrix-binding integrins from both cell compartments in the nonhuman primate. This study is the first to examine the conditions underlying integrin loss from these cell-types during ischemia in vitro and their relation to the changes in vivo. Methods— The impact of normoxia or standardized oxygen-glucose deprivation on integrin expression by murine primary cerebral endothelial cells and astrocytes grown on matrix substrates (collagen IV, laminin, and perlecan) of the basal lamina were quantitatively assessed by flow cytometry. Results— Endothelial cell expression of the &bgr;1 and &agr;5 subunits significantly increased on all matrix ligands, whereas astrocytes displayed modest significant decreases in &agr;5 and &agr;6 subunits. Oxygen-glucose deprivation produced a further significant increase in subunit &bgr;1 expression by both cell types, but a clear decrease in both &agr;1 and &agr;6 subunits by murine astrocytes. Conclusions— Ischemia in vitro significantly increased endothelial cell &bgr;1 expression, which is consistent with the increase in &bgr;1 transcription by microvessels peripheral to the ischemic core. The loss of &agr;1 and &agr;6 integrins from murine astrocytes is identical to that seen in the nonhuman primate in vivo. These findings establish both isolated murine cerebral endothelial cells and astrocytes as potential integrin response cognates of microvascular cells of the neurovascular unit in primates, and allow determination of the mechanisms of their changes to ischemia.

Fibronectin promotes brain capillary endothelial cell survival and proliferation through α5β1 and αvβ3 integrins via MAP kinase signalling

We showed previously that blood vessel maturation in the CNS is associated with a developmental switch in brain capillary endothelial cells (BCEC), from fibronectin signalling during angiogenesis to laminin signalling in the adult. To investigate the functional significance of this switch, we have examined the response of BCEC to different extracellular matrix (ECM) proteins. This showed that BCEC proliferation was significantly promoted by fibronectin (28.2 ± 4.0%) and by vitronectin (14.8 ± 2.1%) compared with uncoated glass (7.2 ± 0.7%), while BCEC survival was significantly promoted by fibronectin (1130 ± 131 cells), vitronectin (830 ± 63 cells), collagen IV (703 ± 77 cells) and laminin (680 ± 34 cells) compared with the uncoated glass (367 ± 48 cells). Biochemical studies showed that BCEC express a limited repertoire of integrins, including the β1 integrins, α3β1, α5β1 and α6β1, and the αvβ3 integrin. Function‐blocking studies showed that the response to fibronectin was mediated equally by the α5β1 and αvβ3 integrins. Analysis of signalling pathways revealed that fibronectin stimulated activation of the p44/p42 MAP kinase signalling pathway and pharmacological inhibitors of this pathway blocked BCEC proliferation on fibronectin. Taken together, these findings show that fibronectin exerts a strong angiogenic influence on endothelial cells (EC) in the CNS, and that this is mediated through the α5β1 and αvβ3 integrins via MAP kinase signalling. In addition to a fundamental role in development, these findings may also have implications in pathological conditions of the CNS where fibronectin is re‐expressed.