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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

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: 1

1-216-444-roles in the CNS.

7929.

E-mail address: [email protected] (R.M. Ransohoff). Pro-inflammatory cytokines, most notably TNF-a and

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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-ahas 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

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 372 8C. Sixteen hours later, nonadherent

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-turer’s protocol. Each analysis was repeated more than

2.1. Reagents three times using RNA from independent astrocyte

cul-tures.

Recombinant human TNF-a was obtained from Becton

Dickinson Labware (Bedford, MA). Recombinant IL-1b 2.5. Immunohistochemistry

was provided by the National Cancer Institute.

Recombi-nant murine IFN-gwas obtained from Gibco BRL (Grand Astrocytes on cover slides were rinsed twice in

phos-Island, NY); recombinant murine SDF-1afrom Peprotech, phate-buffered saline (PBS) and fixed in a solution of

Inc. (Rocky Hill, NJ); goat-anti-CXCR4 and rabbit-anti- methanol: acetone (1:1) for 2 min. After two washes with

Erk2 MAP kinase from Santa Cruz Biotechnology, Inc. TBST [10 mM Tris HCI (pH 8.0), 150 mM NaCl, 0.02%

(Santa Cruz, CA); anti-phospho-Erk1 / 2 MAP kinase Tween-20], nonspecific protein adsorption was blocked by

monoclonal antibody from New England BioLabs Inc.; incubation of cells for 40 min in TBST containing bovine

actinomycin D (ActD), Poly-D-Lysine (PDL), lipopolysac- serum albumin (3%). Polyclonal anti-CXCR4 was

incu-charide (LPS) from Sigma Chemical Co. (St. Louis, MO); bated with the fixed cells for 2 h at room temperature.

Texas red-conjugated anti-goat antibodies from Jackson Cells were washed in TBST, and Texas red-conjugated

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added to the cells for 40 min at room temperature. After probes. As shown in Fig. 1, untreated astrocytes expressed

final wash in TBST, the cells were mounted in Vectashield abundant CXCR4 (Figs. 1–5). Other chemokine receptors

Mounting Medium (Vector Laboratories, Inc., Burlingame, including CXCR2 and BRL-1 remained undetectable by

CA) and examined by fluorescence microscopy. this assay. TNF-atreatment significantly suppressed levels

of CXCR4 mRNA by about 80% (Fig. 1) (mean6S.D. of

2.6. SDS–PAGE and Western blotting CXCR4 / L32 ratios from 5 independent experiments:

2TNF-a, 41.464.6; 1TNF-a, 10.263.7; P,0.0001,

Cells (2–3310 ) were harvested and lysed for 10 min in paired t test). Similar results were observed in cells after

ice-cold lysis buffer [0.5% NP-40, 10% glycerol, 50 mM treatment with LPS, IL-1b (Fig. 2) and IFN-g (data not

Tris–Cl (pH 8.0), 400 mM NaCl, 0.1 mM EDTA, 1 mM shown). Dose–response experiments demonstrated that

sodium orthovanadate, 0.5 mM phenylmethanesulfonyl evident inhibition of CXCR4 mRNA was observed in

fluoride, 1 mM dithiothreitol, 2 mg / ml aprotinin, 2 mg / ml astrocytes treated with 0.1 ng / ml TNF-a (Fig. 3),

imply-leupeptin and 1 mg / ml pepstatin A]. After centrifugation ing that this effect of TNF-a could occur under

physio-for 5 min at 48C in a microfuge, the supernatant solution logic conditions. Kinetic analysis of CXCR4 mRNA

was collected and protein concentration was determined expression revealed that significant inhibition of CXCR4

using Bio-Rad Protein Assay (Bio-Rad laboratories, Her- mRNA accumulation was observed after TNF-a treatment

cules, CA). Equal amount of protein for each sample was for 1 h and was most pronounced after treatment for 24 h

mixed with 23 SDS loading buffer and separated on a (Fig. 4). TNF-a did not stimulate or modulate CXCR4

10% polyacrylamide–SDS gel. Proteins were transferred expression in either mouse fibroblasts or hematopoietic cell

electrophoretically to Immobilon TM PVDF (Millipore) lines used in these experiments, but IFN-g suppressed

membranes. Membranes were blocked with 5% bovine CXCR4 mRNA accumulation in RAW264.7 cells (data not

serum albumin (BSA) (Fraction V) in TBST [10 mM shown), suggesting that the effects of cytokines on

Tris–Cl (pH 7.4), 75 mM NaCl, 1 mM EDTA, 0.1% chemokine receptor expression are cell-type specific.

Tween 20] for at least 1 h at room temperature, incubated To address the mechanism by which TNF-a induced

with the anti-phospho-Erk1 / 2 monoclonal antibody for 1 CXCR4 mRNA expression, the effect of TNF-a on

h, washed in TBST, incubated for 1 h with peroxidase- CXCR4 mRNA stability was examined. Astrocytes were

conjugated second antibody and re-washed in TBST. incubated with medium alone or with TNF-afor 12 h and

Bands were visualized by enhanced chemiluminescence, then ActD was added for an additional 8 h. RNA was

with Renaissance Kit reagents (ECL, Dupont). After anti- isolated at serial time points and examined for level of

phospho-Erk1 / 2 immunoblotting, membranes were strip-ped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM

Tris–HCl (pH 6.8) at 508C for 30 min, neutralized in

TBST and incubated with rabbit-anti-Erk2 antibodies. For statistical analysis, densitometry of p-Erk2 and Erk2 from 4 independent experiments was performed with NIH Image

analysis (v1.65) and the result is shown as mean6S.D.

3. Results

3.1. TNF-a down-regulates CXCR4 expression in

primary astrocytes

Chemokines and their cognate receptors play important roles in manifold inflammatory processes [21,34–36]. Astrocytes participate in the pathophysiology of CNS inflammatory diseases. Previously, we and others reported

that primary mouse astrocytes expressed CXCR4

[12,17,48]. However, regulation of CXCR4 expression has

not been characterized in this cell type. TNF-a among

other pro-inflammatory cytokines is elevated and has pronounced effects in CNS inflammation [39,40,52]. To

Fig. 1. TNF-atreatment reduces steady-state CXCR4 mRNA levels in

evaluate the regulation of CXCR4 on astrocytes, we

primary astrocytes. Murine primary astrocytes were incubated with

treated primary astrocyte cultures with TNF-a, and moni- _{medium alone (lane 1) or TNF-}_a_{(lane 2) for 8 h. RNA was analyzed by}

tored the steady state of CXCR mRNA level by RNase RPA for CXCR, L32 and GAPDH. Data shown are representative of four

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Fig. 2. IL-1b or LPS reduces steady-state CXCR4 mRNA levels in primary astrocytes. Murine primary astrocytes were incubated with medium alone (lane 1) or IL-1b(lane 2), or LPS (100 ng / ml; lane 3) for 8 h. RNA was analyzed by RPA for CXCR, L32 and GAPDH. Data shown are representative of four experiments.

CXCR4 mRNA. The t1 / 2 of CXCR4 mRNA from

un-treated astrocytes was about 2 h, and that of CXCR4

mRNA from TNF-atreated cells was comparable (Fig. 5).

This data indicates that TNF-atreatment does not alter the stability of CXCR4 mRNA, suggesting an effect at the transcriptional level.

3.2. CXCR4 protein expression in primary astrocytes is

reduced by TNF-a

CXCR4 protein expression was evaluated by immuno-histochemistry, to ascertain that there was a predictable relationship between accumulation of CXCR4 mRNA and levels of cognate protein. As demonstrated in Fig. 6, primary astrocytes displayed strong staining with

anti-Fig. 3. Dose-dependent inhibition of CXCR4 mRNA expression by

CXCR4 in the perinuclear cytoplasm, but there was a

TNF-ain murine primary astrocytes. (A): Murine primary astrocytes were

significant reduction of fluorescence intensity of cells after _{incubated with medium alone (lane 1) or with indicated concentration of}

incubation with TNF-a for 48 h, demonstrating that TNF-a (0.1–100 ng / ml) for 16 h (lanes 2–6). RNA was analyzed by

reduced CXCR4 mRNA expression results in decreased RPA for CXCR, L32 and GAPDH. Data shown are representative of three

experiments. (B): Quantitation of the data in (A) by phosphoImager

CXCR4 protein expression.

analysis. Data shown are representative of three experiments (A) and are expressed as mean S.D. (B).

3.3. Effects of TNF-a on MAP kinase activation induced

by SDF-1a in murine primary astrocytes

a for 48 h and then evaluated for Erk1 / 2 MAP kinase

To assess the functional relevance of CXCR4 down- phosphorylation in response to SDF-1a. Western blot

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Fig. 4. TNF-areduces steady-state CXCR4 mRNA levels in murine primary astrocytes: kinetic analysis. (A) Murine primary astrocytes were incubated with medium alone (lane 1) or with TNF-a(10 ng / ml) for various time periods (0.5–24 h; lanes 2–8). RNA was analyzed by RPA for CXCR, L32 and GAPDH. Data shown are representative of three experiments. (B) Quantitation of the data in (A) by phosphoImager analysis. Data represent four independent experiments.

MAP kinase monoclonal antibodies showed that SDF-1a and Erk2 phosphorylation stimulated by SDF-1a (Fig. 7,

stimulated Erk1 / 2 phosphorylation in primary murine lane 4) (mean6S.D. of p-Erk2 / Erk2 ratios from 4

in-astrocytes (Fig. 7, lane 2). TNF-atreatment alone did not dependent experiments: SDF-1a, 3.7560.96; SDF-1a1

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Fig. 5. Half-life of CXCR4 mRNA in murine primary astrocytes. Murine primary astrocytes were incubated with actinomycin D (2.5mg / ml) for the indicated times, and RNA was subjected to RPA. CXCR4 abundance was normalized to L32 for each sample. CXCR4 mRNA at time zero (before the addition of Act-D) was plotted as 100%. A shorter exposure of L32 and GAPDH used for normalization is shown on the bottom. Data shown are representative of three experiments.

anti-Erk2 Western blot demonstrated equal quantities of 4. Discussion

protein in each sample, indicating that TNF-a did not

decrease levels of Erk2 protein. This result supports the Chemokines participate in immune regulation and are

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ceptors is not limited to cells of the hematopoietic system. Of particular interest is the expression of chemokine receptors by neuroepithelial elements, including subpopu-lations of neurons, astrocytes, microglia and oligoden-drocyte progenitors [8]. Specifically, expression of CXCR4 was detected in both neurons and astrocytes in vivo and in vitro [12,17,19,20,48]. CXCR4-null mice exhibited defec-tive cerebellar organogenesis, confirming the physiological significance of chemokine receptor expression in the CNS [55]. HIVgp120 protein-induced neurotoxicity via binding to CXCR4 has been proposed as a pathogenetic event in AIDS-associated neurologic disorders [14,15]. Therefore, studies on CXCR4 expression and regulation in neuro-epithelial cells is fundamental for understanding its physio-pathologic roles in the CNS. In this study, we explored CXC chemokine receptor expression and regulation in primary mouse astrocytes. Our results demonstrate for the

Fig. 6. TNF-a decreases CXCR4 protein expression in primary

as-first time that TNF-a down-regulates CXCR4 expression

trocytes. Murine primary astrocytes were maintained in medium alone (A,

A9) or with TNF-a(B, B9) for 48 h. Cells were stained with anti-CXCR4 in primary astrocytes. Decreases in CXCR4 expression

(A, A9 and B, B9) as described in Materials and methods. A and B _{were functionally relevant since TNF-}_a_-stimulated as-represent fluorescence photomicrographs. Corresponding phase-contrast

trocytes displayed reduced Erk1 / 2 MAP kinase

phos-images are shown in A9and B9(3200). Data shown are representative

phorylation in response to SDF-1a.

of five experiments.

Regulation of chemokine receptor expression has been intensively studied in cells of the hematopoietic system because of their fundamental roles in the immune system

cellular responses to chemokines are dictated by their _{and the finding that they serve as co-receptors for HIV}

cognate receptors expressed on target cells [21,38]. There _{infection. Recent reports have documented regulation of}

is increasing evidence that expression of chemokine re- _{chemokine receptor expression by mediators, most notably}

cytokines [2,10,33,51]. TNF-ais a pro-inflammatory

cyto-kine produced mainly by activated macrophages and astrocytes during HIV infection and other

immune-me-diated inflammatory disorders of the CNS [39,52]. TNF-a

regulates expression of chemokine receptors differentially,

according to the cell type. For example, TNF-a inhibited

CXCR2 not CXCR4 expression in human

polymorphonu-clear leukocytes [2]. Conversely TNF-a stimulated

CXCR4 gene transcription in the promyelocytic cell line U937 and exerted biphasic effects on CXCR4 transcription in human endothelial cells [10]. Importantly, we and others

have documented that astrocytes express TNF-a receptors

and TNF-a modulates CNS inflammation in part via

regulation of cytokine, chemokine, adhesion molecule and

MHC antigen expression by astrocytes

[9,11,23,24,31,32,39,44]. Here, we report that TNF-a

suppressed CXCR4 expression in primary astrocytes.

Other pro-inflammatory stimuli, IL-1b and LPS share

biologic functions with TNF-a and displayed a similar

inhibitory effect on CXCR4 expression in primary

as-Fig. 7. TNF-a decreases SDF-1-stimulated Erk1 / 2 MAP kinase

phos-trocytes. Growth factors EGF, PDGF and TGF-b1, which

phorylation in primary astrocytes. Murine primary astrocytes were left

act on astrocytes, were inert (data not shown). TNF-a

untreated (lane 1) or treated with SDF-1a(100 ng / ml, lane 2) for 10 min,

TNF-afor 48 h (10 ng / ml, lane 3) or pretreated with TNF-a(10 ng / ml) caused a prolonged and pronounced down-regulation of

for 48 h followed SDF-1afor 10 min (100 ng / ml, lane 4) prior to cell _{CXCR4 mRNA in a concentration- and time-dependent} lysis. Erk1 / 2 MAP kinase was analyzed by Western blotting with specific _{manner (Figs. 3 and 4). TNF-}_a_{has been shown to have a} anti-phospho-Erk1 / 2 antibody. The positions of Erk1 / 2 are indicated on

unique time-dependent biphasic effect on CXCR4

expres-the right and molecular weight standards are indicated on expres-the left. After

sion in human endothelial cells [10]. This effect was

stripping, the same membrane was probed with antisera to Erk2. Data

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subsequent reversal and increase in the steady state levels signaling. SDF-1a activated Erk1 / 2 MAP kinase in

pri-of CXCR4 mRNA despite continuous exposure pri-of human mary astrocytes (Fig. 7). Inhibition of CXCR4 mRNA

endothelial cells to TNF-a. Both human endothelial cells accumulation resulted in reduced MAP kinase activation in

and astrocytes are non-hematopoietic cells and exhibit a response to SDF-1a, demonstrating functional relevance.

CXCR4 mRNA half-life of about 2 h (Fig. 5). However, Given these results, it is possible that down-regulation of

the effect of TNF-a on CXCR4 mRNA expression in CXCR4 expression could contribute to anti-tumor effects

astrocytes is different from that in human endothelial cells mediated by TNF-a. TNF-aregulation of CXCR4

expres-since TNF-a exerts a time-dependent, sustained inhibition sion in other neuroepithelial cells and suppression of

of CXCR4 mRNA accumulation (Fig. 4). In murine glioblastoma tumor cell proliferation via inhibition of

astrocytes, TNF-a treatment for 48 h almost completely CXCR4 expression is currently under investigation.

De-abolished CXCR4 mRNA expression (data not shown). tailed understanding of roles and mechanisms of cytokines

Further, the effect of TNF-a on CXCR4 expression was in regulation of chemokine receptor expression by neural

relatively selective and cell-type dependent since the level cells may promote the development of focused alternative

of CCR1 and other CXCR mRNA in astrocytes was not immune-modulating therapeutic strategies for

inflamma-affected by TNF-a treatment [12] and no CXCR4 expres- tory and neoplastic neurologic disorders.

sion was regulated in either mouse fibroblasts or hemato-poietic cell lines (Fig. 1 and data not shown) [12].

Regulation of chemokine receptor expression at the _{Acknowledgements}

levels of mRNA stability has been documented [33,45].

AU-rich elements in the 39-untranslated region (UTR) of _{This research was supported by the National Institutes of}

chemokine receptor mRNA have been proposed to contrib- _{Health (RO1-NS 32151; PO1-CA 62220) and by the}

ute to both positive and negative regulation of chemokine _{Williams Family Fund for Multiple Sclerosis Research.}

receptor mRNA stability. In human monocytes, IFN-g or

LPS down-regulated CCR2 expression by destabilizing the mRNA [20,45]. IL-10 augmented the expression of

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(1)

MAP kinase monoclonal antibodies showed that SDF-1

a

and Erk2 phosphorylation stimulated by SDF-1

a

(Fig. 7,

stimulated Erk1 / 2 phosphorylation in primary murine

lane 4) (mean

6 S.D. of p-Erk2 / Erk2 ratios from 4

in-astrocytes (Fig. 7, lane 2). TNF-

a

treatment alone did not

dependent experiments: SDF-1

a

, 3.75

6 0.96; SDF-1

a1

(2)

anti-Erk2 Western blot demonstrated equal quantities of

4. Discussion

protein in each sample, indicating that TNF-

a

did not

decrease levels of Erk2 protein. This result supports the

Chemokines participate in immune regulation and are

functional significance of CXCR4 inhibition by TNF-

a

.

involved in diverse inflammatory disorders [21,34,35]. The

(3)

ceptors is not limited to cells of the hematopoietic system.

Of particular interest is the expression of chemokine

receptors by neuroepithelial elements, including

subpopu-lations of neurons, astrocytes, microglia and

oligoden-drocyte progenitors [8]. Specifically, expression of CXCR4

was detected in both neurons and astrocytes in vivo and in

vitro [12,17,19,20,48]. CXCR4-null mice exhibited

defec-tive cerebellar organogenesis, confirming the physiological

significance of chemokine receptor expression in the CNS

[55]. HIVgp120 protein-induced neurotoxicity via binding

to CXCR4 has been proposed as a pathogenetic event in

AIDS-associated neurologic disorders [14,15]. Therefore,

studies on CXCR4 expression and regulation in

neuro-epithelial cells is fundamental for understanding its

physio-pathologic roles in the CNS. In this study, we explored

CXC chemokine receptor expression and regulation in

primary mouse astrocytes. Our results demonstrate for the

Fig. 6. TNF-a decreases CXCR4 protein expression in primary

as-first time that TNF-

a

down-regulates CXCR4 expression

trocytes. Murine primary astrocytes were maintained in medium alone (A,

A9) or with TNF-a(B, B9) for 48 h. Cells were stained with anti-CXCR4

_{in primary astrocytes. Decreases in CXCR4 expression}

(A, A9 and B, B9) as described in Materials and methods. A and B

_{were functionally relevant since TNF-}

_a

_-stimulated

as-represent fluorescence photomicrographs. Corresponding phase-contrast

trocytes displayed reduced Erk1 / 2 MAP kinase

phos-images are shown in A9and B9(3200). Data shown are representative

phorylation in response to SDF-1

a

.

of five experiments.

Regulation of chemokine receptor expression has been

intensively studied in cells of the hematopoietic system

because of their fundamental roles in the immune system

cellular responses to chemokines are dictated by their

_{and the finding that they serve as co-receptors for HIV}

cognate receptors expressed on target cells [21,38]. There

_{infection. Recent reports have documented regulation of}

is increasing evidence that expression of chemokine re-

_{chemokine receptor expression by mediators, most notably}

cytokines [2,10,33,51]. TNF-

a

is a pro-inflammatory

cyto-kine produced mainly by activated macrophages and

astrocytes during HIV infection and other

immune-me-diated inflammatory disorders of the CNS [39,52]. TNF-

a

regulates expression of chemokine receptors differentially,

according to the cell type. For example, TNF-

a

inhibited

CXCR2 not CXCR4 expression in human

polymorphonu-clear

leukocytes

[2].

Conversely

TNF-

a

stimulated

CXCR4 gene transcription in the promyelocytic cell line

U937 and exerted biphasic effects on CXCR4 transcription

in human endothelial cells [10]. Importantly, we and others

have documented that astrocytes express TNF-

a

receptors

and TNF-

a

modulates CNS inflammation in part via

regulation of cytokine, chemokine, adhesion molecule and

MHC

antigen

expression

by

astrocytes

[9,11,23,24,31,32,39,44]. Here, we report that TNF-

a

suppressed CXCR4 expression in primary astrocytes.

Other pro-inflammatory stimuli, IL-1

b

and LPS share

biologic functions with TNF-

a

and displayed a similar

inhibitory effect on CXCR4 expression in primary

as-Fig. 7. TNF-a decreases SDF-1-stimulated Erk1 / 2 MAP kinase

phos-trocytes. Growth factors EGF, PDGF and TGF-

b

1, which

phorylation in primary astrocytes. Murine primary astrocytes were left

act on astrocytes, were inert (data not shown). TNF-

a

untreated (lane 1) or treated with SDF-1a(100 ng / ml, lane 2) for 10 min,

TNF-afor 48 h (10 ng / ml, lane 3) or pretreated with TNF-a(10 ng / ml)

caused a prolonged and pronounced down-regulation of

for 48 h followed SDF-1afor 10 min (100 ng / ml, lane 4) prior to cell

_{CXCR4 mRNA in a concentration- and time-dependent}

lysis. Erk1 / 2 MAP kinase was analyzed by Western blotting with specific

_{manner (Figs. 3 and 4). TNF-}

_a

_{has been shown to have a}

anti-phospho-Erk1 / 2 antibody. The positions of Erk1 / 2 are indicated on

unique time-dependent biphasic effect on CXCR4

expres-the right and molecular weight standards are indicated on expres-the left. After

sion in human endothelial cells [10]. This effect was

stripping, the same membrane was probed with antisera to Erk2. Data

(4)

subsequent reversal and increase in the steady state levels

signaling. SDF-1

a

activated Erk1 / 2 MAP kinase in

pri-of CXCR4 mRNA despite continuous exposure pri-of human

mary astrocytes (Fig. 7). Inhibition of CXCR4 mRNA

endothelial cells to TNF-

a

. Both human endothelial cells

accumulation resulted in reduced MAP kinase activation in

and astrocytes are non-hematopoietic cells and exhibit a

response to SDF-1

a

, demonstrating functional relevance.

CXCR4 mRNA half-life of about 2 h (Fig. 5). However,

Given these results, it is possible that down-regulation of

the effect of TNF-

a

on CXCR4 mRNA expression in

CXCR4 expression could contribute to anti-tumor effects

astrocytes is different from that in human endothelial cells

mediated by TNF-

a

. TNF-

a

regulation of CXCR4

expres-since TNF-

a

exerts a time-dependent, sustained inhibition

sion in other neuroepithelial cells and suppression of

of CXCR4 mRNA accumulation (Fig. 4). In murine

glioblastoma tumor cell proliferation via inhibition of

astrocytes, TNF-

a

treatment for 48 h almost completely

CXCR4 expression is currently under investigation.

De-abolished CXCR4 mRNA expression (data not shown).

tailed understanding of roles and mechanisms of cytokines

Further, the effect of TNF-

a

on CXCR4 expression was

in regulation of chemokine receptor expression by neural

relatively selective and cell-type dependent since the level

cells may promote the development of focused alternative

of CCR1 and other CXCR mRNA in astrocytes was not

immune-modulating therapeutic strategies for

inflamma-affected by TNF-

a

treatment [12] and no CXCR4 expres-

tory and neoplastic neurologic disorders.

sion was regulated in either mouse fibroblasts or

hemato-poietic cell lines (Fig. 1 and data not shown) [12].

Regulation of chemokine receptor expression at the

_{Acknowledgements}

levels of mRNA stability has been documented [33,45].

AU-rich elements in the 3

9 -untranslated region (UTR) of

_{This research was supported by the National Institutes of}

chemokine receptor mRNA have been proposed to contrib-

_{Health (RO1-NS 32151; PO1-CA 62220) and by the}

ute to both positive and negative regulation of chemokine

_{Williams Family Fund for Multiple Sclerosis Research.}

receptor mRNA stability. In human monocytes, IFN-

g

or

LPS down-regulated CCR2 expression by destabilizing the

mRNA [20,45]. IL-10 augmented the expression of

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