• Regulation of glial RANTES chemokine expression

      Li, Qingdi Quentin; Bever, Christopher T., Jr.; Burt, David R. (2000)
      Inflammatory tissue injury is an important part of the pathogenesis of multiple sclerosis (MS) and may play a role in such diverse diseases as Alzheimer's disease and spinal cord injury. Glial cells are active participants in the inflammatory response in the central nervous system (CNS), and have been shown to respond to and produce a number of cytokines and chemokines in vivo and in vitro. RANTES (regulated upon activation, normal T-cell expressed and secreted) is a beta-family proinflammatory chemokine that manifests classic chemoattractant activity for lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the mechanism of regulation of RANTES secretion from glial cells is unknown. Therefore, we conducted this study to assess the effect of various biological and pharmacological agents on RANTES expression in a model system for human astrocytes. We show that treatment of the human U-251 MG astrocytic cells, in vitro, with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1beta) stimulates increases in the levels of RANTES protein and mRNA, and the rate of RANTES gene transcription in both a time- and dose-dependent manner. Interferon-gamma (IFN-gamma), an agent shown to have a negative effect on MS patients by stimulating exacerbations, acted synergistically with TNF-alpha or IL-1beta in the induction of RANTES expression in human astrocytic cells. This effect of IFN-gamma was attributable to increased transcription as measured by in vitro nuclear transcript elongation ("run-on") assays and a 50--70% increase in RANTES mRNA half-life. In addition, we used this model system to examine the activity of glatiramer acetate, a drug recently approved for use in the treatment of MS patients, and found that it inhibits TNF-alpha and IL-1beta induced increases in RANTES mRNA and protein levels. We further investigated the mechanisms involved and showed that the inhibitory effect of glatiramer acetate on RANTES expression in these cells is regulated at both the transcriptional and post-transcriptional levels. Finally, we demonstrated that NF-kappaB may be the transcriptional activator responsible for the TNF-alpha and IL-1beta-mediated RANTES gene expression in this system. Our data indicated that TNF-alpha and IL-1beta-induced increases in RANTES mRNA and chemokine were blocked by the NF-kappaB inhibitors gliotoxin, isohelemin, and pyrrolidine dithiocarbamate (PDTC). Electrophoretic mobility shift assays of nuclear extracts from TNF-alpha or IL-1beta-treated cells revealed an increase in DNA-binding activity specific for the NF-kappaB binding site, in the 5'-flanking promoter region of the human RANTES gene. p50 and p65 proteins were confirmed to be the components of the activated NF-kappaB transcription factor complex by supershift analysis. Furthermore, our experiments showed that the increase in NF-kappaB binding activity was prevented by pretreatment with glatiramer acetate or the NF-kappaB inhibitors. These findings suggest that glatiramer acetate may exert its suppressive effect on TNF-alpha and IL-1beta-induced expression of the RANTES chemokine gene, in glial cells, by interfering with NF-kappaB activation. Understanding the mechanism of action of these agents is crucial both in understanding the pathogenesis of MS and in developing novel and useful treatment strategies.
    • Structural and functional analysis of the β-chemokine RANTES: Proposal of a three-site binding hypothesis

      Burns, Jennifer M.; Lewis, George K., Ph.D.; DeVico, Anthony L. (1999)
      Chemokines comprise a family of low molecular weight proteins involved in a variety of biological activities including the activation and regulation of immune responses. A subset of chemokines, including Regulated Upon Activation Normal T Cell Expressed and Secreted (RANTES), Macrophage Inflammatory Protein (MIP)-1alpha, MIP-1beta and Macrophage Derived Chemokine (MDC), block HIV-1 entry of susceptible cells. The anti-viral activity of chemokines is dependent upon binding to 7-transmembrane (TM) G protein-coupled receptors, which also serve as receptors for HIV-1 viral binding to cells. In addition to this dependence upon protein receptors we have demonstrated a reliance of chemokine function on cell surface glycosaminoglycans (GAGs). Through the use of an anti-RANTES monoclonal antibody developed in our laboratory, mAb 4A12, residues in the C-terminal region of RANTES have been identified as those residues critical for GAG binding. Preincubation of mAb 4A12 with RANTES blocks RANTES induced Ca2+ mobilization, inhibits RANTES binding to the cell surface and reverses the HIV-1 anti-viral activity of this chemokine, thus implicating the dependence of chemokine function on cell surface GAGs. Additionally, enzymatic removal of cell surface GAGs from PBMC inhibits the ability of RANTES to mobilize Ca2+ in these cells. Further implication of the importance of GAGs in chemokine function is provided by studies in which cells inherently expressing low levels of cell surface GAGs exhibit reduced ability to mobilize Ca2+ in response to RANTES. Finally, adding back GAGs as soluble complexes to chemokines does not restore chemokine function. Soluble RANTES/GAG complexes are unable to induce Ca2+ mobilization or chemotaxis in susceptible cells; yet they retain HIV-1 anti-viral properties. In conclusion, our data suggest that the current model describing chemokine interactions with protein receptors needs to be extended to include the GAG interaction necessary for functional chemokine binding. Additionally, our findings suggest soluble chemokine/GAG complexes represent 7-TM receptor ligands that no longer elicit the potentially deleterious effects of cell signaling through Ca2+ mobilization and chemotaxis, yet retain their anti-viral activity and therefore represent a potential therapeutic formulation to treat HIV-1 infection.