[期刊论文]

出版年：2001

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-γ-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-α, IL-1β, and IFN-γ. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-α and IL-1β. The peak of CXCR4 expression in response to TNF-α and IL-1β was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-α and IL-1β (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1α (SDF-1α), resulted in an elevation in intracellular Ca²⁺ concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1α treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-γ-inducible protein-10. SDF-1α-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation. Chemokines are small molecular mass (7–14 kDa) proteins that mediate the recruitment and activation of leukocytes and other cells to sites of inflammation, and many other immune responses (for review, see Refs. 1 , 2 , 3 ). Chemokines have been classified into four groups based on the arrangement of the first two of four conserved cysteine residues. In the CXC chemokine family, one amino acid separates the first two cysteine residues. Members of the CXC family include IL-8; IFN-γ-inducible protein, 10 kDa (IP-10) ³ ; growth-related oncogene (GRO-α), β, and γ; monokine-induced by IFN-γ; and stromal cell-derived factor (SDF)-1α and β. CXC chemokines have been classically described as neutrophil chemoattractants, and IP-10 and SDF-1 are involved in T cell activation. The NH₂ terminus of the majority of CXC chemokines contains three amino acid residues (Glu-Leu-Arg: the ERL motif), which precede the first cysteine residue of the primary structure of these chemokines. Members with this ERL motif (ERL⁺) are potent inducers of angiogenesis (for review, see Ref. 4 ). The CC chemokine family is characterized by the first two cysteine residues being adjacent to each other, and include macrophage-inflammatory protein (MIP)-1α, MIP-1β, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, RANTES, eotaxin, and thymus- and activation-regulated chemokine. CC chemokines serve as chemoattractants for monocytes/macrophages, activated T cells, B cells, eosinophils, basophils, and dendritic cells (for review, see Ref. 3 ). Two other chemokine families have recently been described. Lymphotactin, a chemoattractant for T cells, lacks two of the four cysteine residues, and is the only member of the C subfamily ( 5 ). Fractalkine (also known as neurotactin) exists in two forms, either as a membrane-bound or soluble glycoprotein in which the first two cysteine residues are separated by three amino acids (CX₃C) ( 6 ). This chemokine functions as a chemoattractant for T cells and monocytes and is highly expressed in brain ( 6 , 7 ). Chemokines are expressed locally in response to inflammatory stimuli and act to recruit leukocytes via their chemoattractant properties and ability to induce integrin activation. Other important physiological functions have been ascribed to chemokines, including growth-regulatory properties, T cell activation, and Th cell polarization ( 3 , 8 , 9 ). In addition, chemokine subfamilies have angiogenic and angiostatic functions. The ERL⁺ CXC chemokines IL-8 and GRO-α, β, and γ are angiogenic factors, while the ERL⁻ CXC chemokines IP-10 and monokine induced by IFN-γ have angiostatic activity (for review, see Ref. 4 ). Aberrant expression of various chemokines has been implicated in contributing to the pathogenesis of neurologic diseases such as multiple sclerosis and HIV-1-associated dementia (for review, see Ref. 10 , 11 , 12 ), as well as in animal models of CNS disease, including experimental allergic encephalomyelitis, mechanical injury/trauma, ischemia, virus-induced demyelination, and SIV-induced encephalitis ( 13 , 14 , 15 , 16 ). Astrocytes, the major glial cell type in the CNS, have been shown to be a predominant source of chemokine production within the diseased brain ( 17 , 18 , 19 ). A growing literature also indicates that astrocytes are activated in vitro to produce chemokines such as RANTES, IL-8, IP-10, MCP-1, MIP-1α, and MIP-1β ( 20 , 21 , 22 , 23 ). Thus, astrocytes serve as an important source of chemokines within the CNS. CXC chemokines bind to seven-transmembrane domain receptors (CXCR1 to CXCR5) that are coupled to heterotrimeric G proteins (for reviews, see Refs. 3 and 24 ). Signaling by CXC chemokines leads to an increase in intracellular calcium, tyrosine phosphorylation, and activation of mitogen-activated protein kinases (MAPKs) ( 25 , 26 , 27 , 28 ). Little is known about the role of CXC chemokines in the CNS, although several CXC chemokine receptors are expressed in the brain; among these is CXCR4 ( 29 , 30 , 31 , 32 , 33 ). CXCR4 is involved in the directional migration of immune cells in response to its exclusive ligand SDF-1. Furthermore, CXCR4 functions as a coreceptor with CD4 in the binding and fusion of HIV-1, specifically interacting with the viral protein gp120 ( 34 , 35 , 36 ). CXCR4 is expressed in the CNS by neurons, astrocytes, endothelial cells, and microglia ( 27 , 30 , 32 , 37 , 38 , 39 , 40 , 41 , 42 ). CXCR4 is overexpressed in astroglioma tumors, and SDF-1 and CXCR4 expression is colocalized when both are expressed ( 43 , 44 , 45 ). Furthermore, SDF-1 and CXCR4 expression increases with increasing tumor grade ( 45 ). Mice with a targeted mutation in CXCR4 or SDF-1 have a defect in migration of cerebellar granule cells, indicating that CXCR4 signaling can mediate migration of neuronal progenitors ( 46 , 47 ). SDF-1 binding to CXCR4 leads to a number of functional effects on cells of the CNS, including calcium mobilization and microglia migration ( 37 , 38 , 39 , 40 , 41 ). In this study, we have examined the expression of CXCR4 by human astroglioma cells in response to a variety of stimuli, including LPS, TNF-α, IL-1β, IFN-γ, IL-6, IL-4, and IL-10. Our results demonstrate that TNF-α or IL-1β treatment of astroglioma cells induces CXCR4 expression at both the mRNA and protein level, leading to increased responsiveness to SDF-1α. To assess the functional importance of CXCR4 expression, astroglioma transfectants were generated that expressed high levels of CXCR4. Stimulation of these cells with SDF-1α led to intracellular Ca²⁺ concentration ([Ca²⁺]_i) mobilization and selective activation of the extracellular signal-regulated kinase 2 (ERK2) MAPK pathway. Importantly, SDF-1α treatment also resulted in the induction of MCP-1, IL-8, and IP-10 chemokine gene expression in CXCR4 stable transfectants. CXCR4-mediated signaling in astroglioma cells provides another pathway for these cells to express chemokines involved in angiogenesis and inflammation.

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