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-
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-g was obtained from Gibco BRL Grand Astrocytes on cover slides were rinsed twice in phos-
Island, NY; recombinant murine SDF-1a from 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
Laboratories. anti-goat antibody 1:500 dilution in blocking buffer was
Y . Han et al. Brain Research 888 2001 1 –10
3
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-a treatment 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,
6
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-a for 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
