Characterization and visualization of [¹²⁵I] stromal cell-derived factor-1α binding to CXCR4 receptors in rat brain and human neuroblastoma cells

Abstract

Stromal cell-Derived Factor-1 (SDF-1α), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [¹²⁵I]SDF-1α binding to CXCR4 were characterized in the adult rat brain. SDF-1α binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [¹²⁵I]SDF-1α on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [¹²⁵I]SDF-1α binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC₅₀ and K_d of [¹²⁵I]SDF-1α binding were respectively 0.6 nM and 0.36 nM. Similar IC₅₀ values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1α binding (30% inhibition at 10⁻⁶ M). In SK-N-SH cells, [¹²⁵I]SDF-1α bound to CXCR4 with a K_d of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1α induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.

Introduction

Chemokines belong to a family of structurally- and functionally-related small proteins (8–10 kDa) that chemoattract and activate immune and non-immune cells both in vivo and in vitro. They are classified into four groups dependent on the number and spacing of their first cysteine residues, CC, CXC, CX₃C and C (Hesselgesser et al., 1997, Hesselgesser and Horuk, 1999).

Stromal cell-Derived Factor-1 (SDF-1) is a CXC chemokine originally isolated from a bone marrow stromal cell line (Tashiro et al., 1993, Nagasawa et al., 1994). Two forms, α and β (68 and 72 amino acids, respectively), generated by alternative splicing from a unique sdf-1 gene, have been identified of which the α form is the most abundant (Shirozu et al., 1995). SDF-1 mRNA has been demonstrated to be expressed in many organs in adult mice including brain, thymus, heart, lung, liver, kidney, spleen, stomach, intestine and bone marrow (Nagasawa et al., 1999). It is also expressed during embryogenesis in brain, liver, heart and bone marrow spindle-shaped stromal cells (McGrath et al., 1999, Nagasawa et al., 1996a). SDF-1 is a chemoattractant for blood cells (Nagasawa et al., 1999). In the brain, SDF-1 was shown to induce the migration of microglial cells and astrocytes (Tanabe et al., 1997).

SDF-1 was found to be the unique ligand for the CXCR4 receptor (Bleul et al., 1996a, Oberlin et al., 1996) which is a G-protein-coupled seven-transmembrane receptor. CXCR4 is expressed in the brain in a variety of cell types including microglia, astrocytes, neurons, and vascular endothelial cells (Wong et al., 1996, Lavi et al., 1997, Lazarini et al., 2000).

CXCR4 mRNA, like SDF-1, is widely expressed during embryogenesis (Nagasawa et al., 1996, Jazin et al., 1997), particularly in neuronal tissue (McGrath et al., 1999). Importantly, CXCR4 and SDF-1 gene knockout mice have an abnormal development of the cerebellum and of the cardiovascular system. These animals die at birth (Ma et al., 1998; Nagasawa et al., 1996a). SDF-1 has also been reported to induce neuronal apoptosis in vitro (Hesselgesser et al., 1998, Kaul and Lipton, 1999).

Moreover, CXCR4 has been identified as coreceptor for T cell line-tropic strains (X4) of human immunodeficiency virus-1 (HIV-1) which in conjunction with CD4, mediates entry of HIV into its target cells (Feng et al., 1996, Oberlin et al., 1996). The brain is one prominent target of HIV infection, where it leads to HIV encephalitis (HIVE) and HIV-associated dementia (HAD). Furthermore, it was shown that antibodies to CXCR4 as well as SDF-1 analogues can block HIV infection in lymphocytes (Hesselgesser et al.,1997; Heveker et al., 1998). This indicates that chemokine receptors may likely have a functional role in the pathogenesis of HIVE and the blockade of this site could be a possible tool for AIDS treatment. Knowledge of the distribution, physiology, and pathology of chemokines and chemokine receptors such as CXCR4 in the brain is fundamental for understanding the pathogenesis of the interaction between HIV and the central nervous system (CNS).

In the present study, we characterized the pharmacological properties of [¹²⁵I]SDF-1α binding to the CXCR4 receptor in adult rat brain and human neuroblastoma cell line SK-N-SH.

Distribution and characterization of SDF-1α receptors was achieved in rat brain sections using imaging techniques and quantitative autoradiography. These techniques, which quantify free receptors, have been previously described for IL-1 receptors in rat and mice brains (Ban et al., 1993a, Crumeyrolle-Arias et al., 1996) and allow a precise determination of affinity and specificity of receptor–ligand interaction.

The CXCR4 coupling system was also studied in human neuroblastoma cell line to appreciate the functionality of this receptor. In these human cells, the intracellular calcium released under SDF-1 stimulation was visualized using real-time fluorescent dye imaging.