Brain Research 888 2001 1–10 www.elsevier.com locate bres
Research report
TNF-a down-regulates CXCR4 expression in primary murine astrocytes
a a
a a,b ,
Yulong Han , Jintang Wang , Tao He , Richard M. Ransohoff
a
Department of Neurosciences , The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
b
Mellen Center for Multiple Sclerosis Treatment and Research , Department of Neurology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
Accepted 29 August 2000
Abstract
CXC chemokine receptor 4 CXCR4 is a co-receptor for human immunodeficiency virus HIV infection and is believed to be involved in the pathogenesis of AIDS-associated neurologic disorders and brain tumors. The physiological roles of CXCR4 in
developmental patterning of the nervous and hematopoietic system; gastrointestinal angiogenesis; and cardiac organogenesis were established by studies in gene-targeted mice. Studies on CXCR4 expression and regulation in neuroepithelial cells are fundamental for
understanding its physiopathologic roles in the central nervous system CNS. We show here that CXCR4 expression by primary mouse astrocytes is suppressed by exposure to tumor necrosis factor-a TNF-a. TNF-a caused a pronounced down-regulation of CXCR4
mRNA in a dose- and time-dependent manner. TNF-a-mediated decrease of CXCR4 mRNA accumulation resulted in decreased CXCR4 protein expression. As a result, the ability of stromal cell-derived factor-1a SDF-1a to induce activation of MAP kinases, Erk1 2 was
impaired. The half life of CXCR4 mRNA in the presence and absence of TNF-a stimulation was comparable, suggesting that TNF-a down-regulated CXCR4 mRNA at the transcriptional level. These results suggest that TNF-a could modulate HIV and brain tumor
pathogenesis and immune-mediated inflammation in the central nervous system CNS by regulation of CXCR4 expression.
2001
Elsevier Science B.V. All rights reserved.
Keywords : Astrocyte; TNF-a; Chemokine receptor; Gene regulation
1. Introduction kines on chemokine receptor expression strictly depend on
cell type as well as the stage of differentiation and Chemokines are small cytokines that mediate directed
maturation of the cell [2,10,33,51]. migration and activation of target cells and act through
CXCR4 serves as a co-receptor for human immuno- specific seven-transmembrane-spanning G-protein-coupled
deficiency virus HIV infection [6]. CXCR4 is expressed receptors [18,21,37,41]. Chemokine and chemokine re-
on both neurons and glial cells, and is believed to be ceptor system has been implicated in diverse human
involved in the pathogenesis of AIDS-associated neuro- diseases [21,41]. Recent results indicate that regulation of
logic disorders [8,16,19,20,47,48,50]. The physiological expression of chemokine receptors is a crucial point for the
roles of CXCR4 in developmental patterning of the regulation of chemokine action and receptor function
nervous and hematopoietic system; gastrointestinal angio- [33,55]. Cytokines are major regulators of chemokine
genesis; and cardiac organogenesis were established by receptor expression. Positive or negative effects of cyto-
studies in gene-targeted mice [22,46,55]. In addition, overexpression of CXCR4 was reported to be required for
proliferation of glioblastoma tumor cells [42,43]. These
Abbreviations: CXCR, CXC chemokine receptor; SDF-1a, stromal
observations indicate that CXCR4 has diverse functions in
cell-derived factor-1a; RPA, RNase protection assay; HIV, human im-
the physiology and pathology of the CNS. Therefore,
munodeficiency virus; CNS, central nervous system; LPS, lipopolysac-
studies on CXCR4 expression and regulation in neuro-
charide; IFN, interferon
epithelial cells is fundamental for understanding its varied
Corresponding author. Tel.: 11-216-444-8939; fax: 11-216-444-
roles in the CNS.
7929. E-mail address
: ransohrccf.org R.M. Ransohoff.
Pro-inflammatory cytokines, most notably TNF-a and
0006-8993 01 – see front matter
2001 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 2 4 - 3
2 Y
IFN-g, produced by inflammatory cells in the CNS, have 2.2. Mice
been proposed to contribute to brain injury or to modulate pathological processes following HIV infection and other
Male SWR and female SJL J mice were purchased from diversity of brain insults [19,40]. TNF-a has potent effects
The Jackson Laboratory Bar Harbor, Maine and were on neuroepithelial cell proliferation, differentiation, adhe-
bred in our animal facilities. All experimental procedures sion, and migration dependent on TNF-a concentration
were approved by the institution’s animal care committee and microenvironment [52]. Such TNF-a effects are at
and were in accordance with the guidelines instituted by least in part via regulation of cytokine, chemokine, adhe-
the Institution of Laboratory Resources, National Research sion molecule and MHC antigen expression. The effect of
Council Department of Health and Human Service, TNF-a exposure on chemokine receptor expression by
National Institutes of Health Publication. neuroepithelial cells has not been well characterized.
Binding studies indicate that multiple chemokines within 2.3. Astrocyte isolation and culture
a subfamily may bind to a single receptor, and multiple receptors may bind a single chemokine [21,37]. But to
Astrocytes were purified from P0-P3 mouse cerebrum date, stromal cell-derived factor-1a SDF-1a is the only
by differential adhesion, as described previously [12]. physiological ligand for CXCR4 [1,3,4,30]. SDF-1a is
Briefly, after removal of the meninges, the cerebra were constitutively expressed in vivo in lymphoid, CNS and
separated into
single-cell suspensions
by mincing,
other tissues of mice and humans [54]. A similar profile of trypsinizing and passage through nylon mesh. The primary
deficiencies is found in CXCR4 and SDF-1a deficient glial cells were washed and then grown in PDL-coated 75
2
mice [22,29,46,55]. Expression of SDF-1a in the CNS is cm
flasks in Dulbecco’s modified Eagle’s medium detected predominantly in astrocytes [53]. Acting through
DMEM; Gibco BRL, supplemented with 10 serum, 2 CXCR4, SDF-1a induces chemotaxis, stimulates calcium
mM glutamine, 50 U ml penicillin and 50 mg ml strep- response and blocks HIV infection [3,6,13,28,30,49]. SDF-
tomycin referred to as complete medium in a moist 5 1a regulation of cell growth is possibly mediated through
CO atmosphere at 378C. Sixteen hours later, nonadherent
2
modulation of signaling molecules and transcription factors oligodendrocytes and microglial cells were removed by
including Erk1 2 MAP kinases, PI-3 kinase, NF-kB and intensively shaking and washing. The purity of the as-
CREB [7,25]. trocyte population was .95, as determined by indirect
Here, we show that expression of CXCR4 by primary immunofluorescence assay with anti-glial fibrillary acidic
mouse astrocytes is down-regulated by TNF-a. TNF-a protein GFAP antibodies.
suppressed CXCR4 mRNA and protein expression and All astrocytes were cultured for a total of 16 to 22 days.
inhibited SDF-1a-stimulated Erk1 2 phosphorylation. Fur- To remove residual oligodendrocytes and microglial cells,
ther, we provide evidence that TNF-a down-regulated the flasks were shaken and washed again before harvesting.
CXCR4 mRNA at the transcriptional level. These results indicate a novel role of TNF-a in modulation of CNS
2.4. RNA isolation and RNase protection assay pathogenesis including HIV infection, immune-mediated
inflammation and brain tumor development. Total cellular RNA, isolated using TRIzol reagent
Gibco BRL, MD, was analyzed by RNase protection assay for mouse CXC chemokine receptors with Multi-
probe Template Sets and In Vitro Transcription Kit
2. Materials and methods PharMingen, San Diego, CA according to the manufac-
