GLIA 22:171–179 (1998)

Transforming Growth Factor–Beta

Inhibition of Cytokine-Induced
Vascular Cell Adhesion Molecule-1
Expression in Human Astrocytes
MARGARET K. WINKLER1,2 AND ETTY N. BENVENISTE1*
1Department of Cell Biology, the University of Alabama at Birmingham, Birmingham, Alabama
2Division of Critical Care Medicine, Department of Pediatrics, the University of Alabama

at Birmingham, Birmingham, Alabama

KEY WORDS cytokines; adhesion molecules; astroglioma cells

ABSTRACT Leukocyte transmigration across the blood-brain barrier (BBB) is a

cardinal feature of central nervous system (CNS) inflammation. Astrocytes form an
integral part, both structurally and functionally, of the BBB. Vascular cell adhesion
molecule-1 (VCAM-1), a member of the immunoglobulin gene superfamily, is involved in
extravasation into inflamed tissues and activation of T-lymphocytes. In this study, we
investigated the role of TGF-b, an immunosuppressive cytokine, in regulating cytokine-
induced VCAM-1 expression in astrocytes. Human astroglioma cell lines and primary
human fetal astrocytes were examined for VCAM-1 gene expression after treatment with
proinflammatory cytokines (TNF-a, IL-1b, IFN-g) in the absence or presence of TGF-b.
Astroglioma cell lines as well as primary human fetal astrocytes expressed low levels of
VCAM-1 constitutively, and the proinflammatory cytokines induced marked increases in
VCAM-1 expression, particularly TNF-a and IL-1b. The inclusion of TGF-b1 or TGF-b2
with the proinflammatory cytokines inhibited VCAM-1 gene expression to varying
degrees (33–93%) in all the astroglioma cell lines and primary fetal cells. These results
indicate that TGF-b is an important regulator of cytokine induced VCAM-1 expression on
astrocytes and may prove useful clinically in controlling CNS inflammation. GLIA
22:171–179, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTION nella et al., 1991; Cannella and Raine, 1995; Nottet et

The central nervous system (CNS) has historically
been characterized as an immune-privileged site. Re-
cent studies have shown that low levels of leukocyte
trafficking in and out of the CNS is a normal event
(Hickey et al., 1991). However, during pathologic condi-
tions in the brain such as multiple sclerosis (MS),
experimental allergic encephalomyelitis (EAE), and
AIDS dementia complex, the number of leukocytes
entering the brain is significantly increased (for review,
see Benveniste, 1995). Cell adhesion molecules such as
intercellular adhesion molecule-1 (ICAM-1) and vascu-
lar cell adhesion molecule-1 (VCAM-1) are increased in
the CNS, particularly during times of inflammation,
and are thought to contribute to extravasation of
leukocytes across the BBB and into CNS parenchyma
(Barten and Ruddle, 1994; Brosnan et al., 1995; Can-
al., 1996). Astrocytes are the most numerous of all glial
cell types within the CNS and are capable of participat-
ing in inflammatory responses by expression of cyto-
kines, chemokines, and adhesion molecules which re-
cruit immunocompetent cells from the peripheral
circulation (for review, see Merrill and Benveniste,
1996). In addition, astrocytic end-feet on CNS capillar-
ies contribute a major structural component of the
BBB, and formation of tight junctions between endothe-
Contract grant sponsor: Multiple Sclerosis Society; Contract grant number:
RG-2269; Contract grant sponsor: National Institutes of Health; Contract grant
numbers: NS29719, NS34856, MH55795.
*Correspondence to: Etty N. Benveniste, Ph.D., Department of Cell Biology,
University of Alabama at Birmingham, Birmingham, Alabama 35294-0005.
E-mail: ebenven@cellbio.bhs.uab.edu
Received 12 March 1997; Accepted 24 June 1997

r 1998 Wiley-Liss, Inc.

172 WINKLER AND BENVENISTE
lial cells is induced by astrocytes contacting the endothe-
lium (Janzer and Raff, 1987).
The proinflammatory cytokines tumor necrosis fac-
tor-a (TNF-a), interleukin-1b (IL-1b), and interferon-g
(IFN-g) induce the expression of ICAM-1 and VCAM-1
on various cell types including endothelial cells, mono-
cytes, lymphocytes, and astrocytes (Bevilacqua et al.,
1994; Frohman et al., 1989; Henninger et al., 1997;
Hurwitz et al., 1992; Rosenman et al., 1995; Shrikant et
al., 1994). VCAM-1 is a member of the immunoglobulin
gene superfamily and is the ligand for very late anti-
gen-4 (VLA-4) which is expressed by leukocytes (Elices
et al., 1990). Interaction of VLA-4 with VCAM-1 plays
an important role in the extravasation of leukocytes out
of the bloodstream into inflamed tissues (for review, see
Carlos and Harlan, 1994). In addition, VCAM-1 is
involved in the initiation of T-cell activation and prolif-
eration, and, ultimately, T-cell death (Damle et al.,
1993; Damle et al., 1992). VLA-4/VCAM-1 interactions
at the BBB may be especially important for leukocyte
entry into the CNS and ensuing autoimmune re-
sponses. Antigen-specific T lymphocytes that do not
express VLA-4 are incapable of crossing the BBB to
initiate inflammatory events (Baron et al., 1993; Kuch-
roo et al., 1993). Therapy with monoclonal antibody
against VLA-4 diminished the infiltration of VLA-4-
positive cells into the brain, and the clinical and
histopathological signs of EAE (Soilu-Hänninen et al.,
1997; Yednock et al., 1992). Anti-TNF-a treatment also
inhibits the incidence and severity of EAE (Ruddle et
al., 1990; Santambrogio et al., 1993), and one mecha-
nism of action is by inhibiting VCAM-1 expression on
spinal cord vessels leading to a significant reduction in
leukocyte entry into the CNS (Barten and Ruddle,
1994). Thus, blocking the interaction of VLA-4-positive
T-cells with VCAM-1-positive endothelial cells or glial
cells may attenuate inflammation within the CNS.
The pleiotropic cytokine transforming growth factor-
beta (TGF-b) is produced by numerous cell types,
including T cells, platelets, macrophages, osteoblasts,
and astrocytes (Morganti-Kossmann et al., 1992; Saad
et al., 1991; Wahl, 1994). There are five known isoforms
of TGF-b, three of which are expressed in humans
(TGF-b1, TGF-b2, and TGF-b3). Of the three, TGF-b1
and TGF-b2 show the greatest degree of homology and
are typically used in most studies. TGF-b is a potent
modulator of immunologic functions by exerting inhibi-
tory effects on the development and differentiation of
immunocompetent cells (for review, see Wahl, 1994).
TGF-b has a number of immunosuppressive effects on
glial cells. For example, TGF-b inhibits class II MHC
and ICAM-1 expression in astrocytes (Panek and Ben-
veniste, 1995; Panek et al., 1995; Shrikant et al., 1996)
and inhibits the production of TNF-a in glial cells
(Benveniste et al., 1994). Mice deficient for the TGF-b1
gene have fatal multifocal inflammatory disease, dem-
onstrating the importance of TGF-b immunoregulatory
properties (Shull et al., 1992). In addition, TGF-b1 de-
ficient mice have increased expression of class II MHC
antigens and ICAM-1 molecules, indicating that one
natural function of TGF-b is to control the expression of
these genes (Diebold et al., 1995; Geiser et al., 1993).
Administration of TGF-b1 or TGF-b2 to animals with
EAE improves their clinical course (Johns et al., 1991;
Kuruvilla et al., 1991; Racke et al., 1991), probably due
to the ability to reduce the accumulation of lymphocytes
in the CNS (Fabry et al., 1995). This effect of TGF-b may
be due to inhibition of adhesion molecule expression.
In this study, we investigated the ability of TGF-b to
modulate VCAM-1 expression in human astroglioma
cell lines and primary human fetal astrocytes. Our
results demonstrate that VCAM-1 is readily inducible
on astroglioma cell lines and primary human fetal
astrocytes after stimulation with TNF-a, IL-1b, and
IFN-g. In addition, cytokine-induced VCAM-1 expres-
sion is significantly inhibited by treatment with TGF-b1
and TGF-b2. These results suggest that TGF-b plays an
important role in decreasing VCAM-1 expression under
inflammatory conditions.
MATERIALS AND METHODS
Astroglioma Cell Lines
U251-MG, D54-MG, and CH235-MG human astro-
glioma cell lines were used in this study (Bethea et al.,
1992; Bethea et al., 1990; Lee and Benveniste, 1996).
All three cell lines are high grade astroglioma cells that
were used in these experiments from passage 6-40,
except for CH235, which was used from passage 68-80.
U251, D54, and CH235 cells were maintained in HAMS
F-12/DMEM media with 2 mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 10% heat inacti-
vated fetal bovine serum. Cells were seeded at 2–4 3
106 cells/dish and grown to confluence in 100 mm
dishes. For passage, monolayers were rinsed with PBS
and then dislodged by trypsinization (0.25% trypsin,
0.02% EDTA).
Primary Human Fetal Astrocytes
Fetal astrocytes were generously provided by Dr.
Eugene Major (National Institutes of Health, Bethesda,
MD). Human fetal astrocytes were prepared as previ-
ously described (Dayton and Major, 1996). Briefly,
human fetal brain tissue between 8 and 15 weeks’
gestation from legal curettage were minced and passed
through a 19 gauge needle. Cells were plated at 1 3 106
cells/ml in Eagle’s minimum essential media with 10%
fetal bovine serum, 2 mM glutamine, penicillin/strepto-
mycin, and fungizone. Cells were passaged for 4–6
weeks prior to use. Astrocyte purity was determined by
staining for glial fibrillary acidic protein (GFAP); the
astrocyte cultures were .98% GFAP positive (Dayton
and Major, 1996).
Reagents
Human recombinant TNF-a (specific activity:
5.6 3 107 U/mg) was the generous gift of Genentech,
 TGF-b INHIBITION OF VCAM-1 EXPRESSION
Inc. (South San Francisco, CA); human recombinant
IL-1b (specific activity: 5 3 108 U/mg) was obtained
from Genzyme Corporation (Cambridge, MA); human
recombinant IFN-g was the generous gift of Biogen
(Cambridge, MA); human recombinant TGF-b2 (lot
CB2010) was a generous gift of Genzyme Corporation
(Cambridge, MA); and human recombinant TGF-b1
was purchased from R&D Systems (Minneapolis, MN).
Anti-human VCAM-1 antibodies (clone 1G11B1, IgG1
isotype) were obtained from Serotec (Washington, DC).
1D.11 antibody (lot 2921-106), which neutralizes all
three mammalian isoforms of TGF-b, and the 3C7
antibody (lot 2921-77), which neutralizes only TGF-b2
and TGF-b3, were generous gifts of the Genzyme
Corporation (Cambridge, MA). Goat anti-mouse H and
L chain FITC-labeled antibodies were from Southern
Biotechnologies (Birmingham, AL).
RNA Isolation
Total cellular RNA was isolated from confluent mono-
layers of the cell lines that were incubated for various
time periods with the different cytokines. RNA isolation
was performed as previously described (Shrikant et al.,
1995). Briefly, cells were washed once with PBS and
lysed directly in the culture dish. RNA was extracted
with guanidinium isothiocyanate and phenol and pre-
cipitated with ethanol.
Riboprobes and RNase Protection Assay (RPA)
A pBluescript SK(6) vector (Stratagene) containing a
fragment of the human VCAM-1 cDNA (bp 1307-2811)
was obtained from the ATCC. The vector was linearized
with Spe1, which digests within the VCAM-1 cDNA
insert. In vitro transcription of this linearized vector
with T7 RNA polymerase results in a 449 bp antisense
RNA probe. The protected fragment is 427 nt. A pAMP-1
vector (GIBCO) containing a fragment of the human
GAPDH cDNA (bp 43-531) was linearized with Nco1,
which digests within the GAPDH cDNA insert. In vitro
transcription of this linearized vector with T7 RNA
polymerase results in a 290 bp antisense RNA probe.
The protected fragment is 230 nt. GAPDH mRNA was
utilized as a ‘‘housekeeping gene,’’ as its levels are not
affected by cytokine treatment.
RNase protection assay (RPA) was carried out with a
RPA kit according to the manufacturer’s instructions
(Ambion, Austin, TX) as previously described (Shrikant
et al., 1995). Briefly, 10 µg of total cellular RNA from the
astroglioma cell lines were hybridized with VCAM-1
(5 3 104 cpm) and GAPDH (2.5 3 104 cpm) riboprobes
at 42°C overnight in 20 µl of 40 mM PIPES pH 6.4, 80%
deionized formamide, 400 mM NaOAc, and 1 mM
EDTA. The hybridized mixture was then treated with
RNase A/T1 (1:500 dilution in 200 µl of RNase digestion
buffer) at room temperature for 45 min. RNA was
precipitated and analyzed by 5% denaturing (8M urea)
173
polyacrylamide gel electrophoresis. The gels were ex-
posed to X-ray film and quantitation of protected RNA
fragments was performed by scanning with the Phosphor-
Imager (Molecular Dynamics, Sunnyvale, CA). Values
for VCAM-1 were normalized to GAPDH mRNA levels
for each experimental condition. Data that is repre-
sented in graphic form is done so as the ratio of
VCAM-1 mRNA to GAPDH mRNA.
Analysis of VCAM-1 Protein Expression by
Immunofluorescence Flow Cytometry
Human astroglioma cell lines or primary human fetal
astrocytes were plated at 5 3 105 in 6-well (35-mm2 )
plates (Costar, Cambridge, MA) and grown to conflu-
ency. Once cells reached confluency, the original me-
dium was aspirated off and fresh serum-free media (1
ml) was added to wells. Duplicate wells of cells were
treated with medium alone, TNF-a, IL-1b, IFN-g, or
one of these cytokines plus TGF-b for various times.
Cells were trypsinized, suspended in PBS containing
5% FBS and 0.02% azide, stained with anti-human
VCAM-1 (1:500 dilution), washed twice, and then
stained with FITC labeled goat anti-mouse antibodies
(1:100 dilution). After washing three times, cells were
fixed in 1% paraformaldehyde and analyzed on the
FACStar (Becton-Dickinson, Mountain View, CA) for
VCAM-1 expression. We have previously determined
that trypsinization does not affect cell surface expres-
sion of VCAM-1 (Rosenman et al., 1995). Negative
controls were incubated with an isotype matched irrel-
evant monoclonal antibody (IgG1, clone 1A29, Pharmin-
gen). Ten thousand cells were analyzed for each sample.
Total VCAM-1 expression is calculated as the percent-
age of positive cells 3 mean fluorescence intensity (MFI).
Statistical Analysis
Levels of significance for comparison between samples
were determined using Student’s t-test distribution.
RESULTS
Cytokine Induction of VCAM-1 Protein
Expression by Astroglioma Cells
We have previously demonstrated that TNF-a and
IFN-g induce VCAM-1 expression in the human astro-
glioma cell lines CRT and STT (Rosenmann et al.,
1995). We extended these observations by investigating
VCAM-1 expression in three other astroglioma cell
lines (U251, CH235, D54) in response to the proinflam-
matory cytokines TNF-a, IL-1b, and IFN-g. Cell lines
were treated for 6, 12, 24, or 36 h with the cytokines,
then VCAM-1 expression was assessed by flow cytome-
try. In U251 cells, VCAM-1 induction by all three
cytokines was evident at 6 h and peaked after 12–24 h
of cytokine exposure (Fig. 1). Comparable results were
174 WINKLER AND BENVENISTE

Fig. 1. TNF-a, IL-1b, and IFN-g induce VCAM-1 protein expression

in the U251-MG human astroglioma cell line. U251-MG cells were
seeded at 5 3 105 cells/well in six-well plates and grown to confluence.
Cells were then washed and placed in serum-free medium for treat-
ment with TNF-a (10 ng/ml), IL-1b (1 ng/ml), or IFN-g (100 U/ml) for
6–36 h. VCAM-1 protein expression was assessed by FACS analysis.
Total VCAM-1 expression is calculated as a product of % positive
cells 3 mean fluorescence intensity. Representative of four experiments.

obtained for the CH235 and D54 cell lines (data not
shown). TNF-a was the strongest inducer of VCAM-1
expression in all three cell lines, followed by IL-1b and
then IFN-g. U251 astroglioma cells expressed the high-
est level of cytokine-induced VCAM-1 expression and
were chosen for further study.

Cytokine Induction of VCAM-1 mRNA

We next examined the effect of proinflammatory

cytokines on VCAM-1 mRNA expression in U251 cells.
TNF-a was chosen as the stimulus since it was the most
potent inducer of VCAM-1 protein expression in these
cells. U251 cells were incubated for 1–8 h with TNF-a
(10 ng/ml). Total RNA was harvested and then evalu-
ated by ribonuclease protection assay (RPA) for VCAM-1
mRNA expression. Unstimulated U251 cells do not
constitutively express VCAM-1 mRNA (Fig. 2A, lane 1);
however, TNF-a stimulation induces VCAM-1 mRNA
in a time dependent manner, with optimal expression
after 8 h of exposure (Fig. 2A, lanes 2–6). Comparable
results were obtained using IL-1b or IFN-g as the
inducing agent, with maximal VCAM-1 mRNA levels
seen at 8 h (data not shown). PhosphorImager quantita-
tion of the blot is shown in Figure 2B.
TGF-b1 Inhibits Cytokine Induced VCAM-1
Protein Expression
To characterize the effect of TGF-b1 on VCAM-1
protein expression, we exposed astroglioma cells to
cotreatment with TGF-b1 and the proinflammatory
cytokines (TNF-a, IL-1b, IFN-g). These experiments
were of interest because human astroglioma cells ex-
press TGF-b1 in vitro and in vivo (Morganti-Kossmann
Fig. 2. TNF-a induces VCAM-1 mRNA expression in U251 cells.
U251 cells were treated with TNF-a (10 ng/ml) for 1, 2, 4, 6, and 8 h.
RNA was extracted and examined by RPA for VCAM-1 and GAPDH
mRNA expression (A). PhosphorImager quantitation of the blot is
shown in (B). Data has been normalized to GAPDH expression and is
represented as a ratio of VCAM-1/GAPDH mRNA. Representative of
three experiments.
et al., 1992; Saad et al., 1991), and this expression is
proposed to be implicated in immune system evasion by
gliomas (Bodmer et al., 1989; Constam et al., 1992;
Horst et al., 1992). U251 cells were treated with the
proinflammatory cytokines and TGF-b1 for 24 h, then
VCAM-1 protein expression was assessed by FACS
analysis. VCAM-1 induction by all three cytokines was
significantly different from the medium control at the
95% confidence level. TGF-b1 (10 ng/ml) alone had no
effect on VCAM-1 protein expression, but strongly
inhibited VCAM-1 protein expression induced by TNF-a,
IL-1b and IFN-g (Table 1). TGF-b1 inhibited cytokine-
induced VCAM-1 protein expression in the CH235 and
D54 cell lines to a similar extent (data not shown).
Previous work has shown that optimal TGF-b1 inhibi-
tion of cytokine-induced ICAM-1 expression in astro-
cytes/astroglioma cells is seen after a 12 h pretreatment
 TGF-b INHIBITION OF VCAM-1 EXPRESSION 175
TABLE 1. TGF-b inhibits cytokine-induced VCAM-1 expression
in U251 cells

% positive Total %
Cell treatment cells MFIa VCAM-1b inhibition
Medium for 24 h 0.67 6 0.2c 24.3 6 8.7 16.3
TGF-b1 (10 ng/ml)
for 24 h 1.23 6 0.3 23.5 6 5.4 28.9
TNF-a (10 ng/ml)
for 24 h 91.7 6 6.3 164.0 6 30.0 15,038.8
TNF-a 1 TGF-b
for 24 h 41.7 6 6.3 27.0 6 14.6 1,125.9* 93%d
IL-1b (1 ng/ml)
for 24 h 75.3 6 15.5 132.8 6 62.2 9,999.8
IL-1b 1 TGF-b
for 24 h 26.6 6 9.1 40.1 6 22.5 1,066.7* 90%e
IFN-g (100 U/ml)
for 24 h 38.9 6 10.1 46.4 6 11.7 1,805.0
IFN-g 1 TGF-b
for 24 h 15.8 6 9.4 18.1 6 5.4 286.0* 85%f
aMFI 5 Mean Fluorescence Intensity.
bTotalVCAM-1 5 % positive cells 3 MFI.
cMean 6 S.D. of three experiments.
d% inhibition compared to TNF-a alone.
e% inhibition compared to IL-1b alone.
f% inhibition compared to IFN-g alone.

*Statistically different from TNF-a, IL-1b, or IFN-g treatment alone at the 95%
confidence level.

with TGF-b1 (Shrikant et al., 1996). However, pretreat-

ment with TGF-b1 for up to 36 h did not enhance the
extent of inhibition of VCAM-1 compared to cotreat-
ment in human astroglioma cell lines (data not shown).

TGF-b1 Inhibits Cytokine Induced VCAM-1

mRNA Expression

To further understand the mechanism by which

TGF-b1 inhibits cytokine-induced VCAM-1 expression,
we evaluated the effect of TGF-b1 on TNF-a induced
VCAM-1 mRNA in U251 cells. Ribonuclease protection
assay was performed on RNA isolated from U251 cells
that were treated with TNF-a, TGF-b1, or both for
varying time intervals. As shown in Figure 3A, VCAM-1
mRNA expression was induced in U251 cells in re-
sponse to TNF-a (lane 3), and TGF-b1 alone had no
effect on VCAM-1 mRNA expression (lane 2). Cotreat-
ment of cells with TNF-a and TGF-b1 for 8 h decreased
the level of VCAM-1 mRNA by ,65% (lane 4). Pretreat-
ment with TGF-b1 for 4, 8, or 12 h prior to TNF-a
treatment did not enhance the extent of VCAM-1
inhibition (lanes 5–7). Quantitation of this blot is
shown in Figure 3B. These results indicate that TGF-b1
acts to inhibit TNF-a induced VCAM-1 mRNA expres-
sion.
TGF-b1 inhibits TNF-a induced VCAM-1 mRNA
expression in a dose dependent manner (Fig. 4). U251
cells were exposed to TNF-a (10 ng/ml) and varying
concentrations of TGF-b1 (0.1–50 ng/ml). Total RNA
was harvested and evaluated by RPA. At a concentra-
tion of 10 ng/ml of TGF-b1, maximal inhibition of
TNF-a induced VCAM-1 was achieved (Fig. 4, lane 5).
Ribonuclease protection assay was also performed on
RNA isolated from U251 cells that were treated with
TNF-a alone or TNF-a plus TGF-b1 for 4, 6, or 8 h (Fig.
5A). The highest levels of VCAM-1 mRNA were seen at
Fig. 3. TGF-b inhibits TNF-a induced VCAM-1 mRNA expression
in U251 cells. U251 cells were treated with TGF-b1 (10 ng/ml) plus
TNF-a (10 ng/ml) for 8 h, or cells were pretreated with TGF-b1 for 4, 8,
or 12 h, then exposed to TNF-a for 8 h. RNA was extracted and
examined by RPA (A). PhosphorImager quantitation of the blot is
shown in (B). Data has been normalized to GAPDH expression and is
represented as a ratio of VCAM-1/GAPDH mRNA. Representative of
three experiments.
8 h after exposure to TNF-a, and inhibition of TNF-a
induced VCAM-1 mRNA by TGF-b1 was also strongest
after an 8 h exposure (Fig. 5A, lanes 6 and 7). Quantita-
tion of the blot is shown in Figure 5B.
Specificity of TGF-b Inhibition of TNF-a Induced
VCAM-1 Expression
To characterize the specificity of TGF-b1 inhibition of
TNF-a induced VCAM-1, we utilized neutralizing anti-
bodies against TGF-b. U251 cells were incubated for
24 h in serum-free medium with TNF-a (10 ng/ml) and
TGF-b1 (10 ng/ml). In addition, either the 1D.11 anti-
176 WINKLER AND BENVENISTE

Fig. 4. TGF-b1 inhibits TNF-a induced VCAM-1 in a dose-

dependent manner. U251 cells were incubated with TNF-a (10 ng/ml)
in the absence or presence of varying concentrations of TGF-b1
(50–0.1 ng/ml) for 8 h (lanes 3–7). RNA was extracted and evaluated by
RPA. Cells were also incubated with TGF-b1 alone (10 ng/ml) for 8 h
(lane 2). Representative of four experiments.

body (which recognizes all three isotypes of TGF-b) or

the 3C7 antibody (which recognizes only TGF-b2 and
b3 isotypes) was added. VCAM-1 protein expression
was assessed by FACS analysis. Figure 6A shows the
results of neutralization of TGF-b1 activity with the
antibodies; TNF-a induced levels of VCAM-1 protein
were inhibited ,76% by TGF-b1, and inclusion of the
1D.11 antibody reversed the inhibitory effect of TGF-
b1, with minimal inhibition observed (,30%). Note
that the 3C7 antibody was not able to neutralize the
inhibitory effect of TGF-b1 on TNF-a induced VCAM-1
protein expression (,82% inhibition). In addition, the
experiments were repeated using TGF-b2 to inhibit
TNF-a induction of VCAM-1 protein (Fig. 6B). TGF-b2
was as effective as TGF-b1 in inhibiting TNF-a induced
VCAM-1 expression (,78%). 1D.11 and 3C7 antibodies
were both able to neutralize the inhibitory effect of
TGF-b2 on TNF-a induced VCAM-1, reducing inhibi-
tion to ,15%.
Regulation of VCAM-1 Expression in Primary
Human Fetal Astrocytes
We assessed the regulation of VCAM-1 expression in
primary human fetal astrocytes to determine if VCAM-1
Fig. 5. Kinetics of TGF-b1 inhibition of TNF-a induced VCAM-1
mRNA expression in U251 cells. U251 cells were incubated in serum-
free medium alone for 8 h (lane 1), or were simultaneously incubated
with TNF-a (10 ng/ml) and TGF-b1 (10 ng/ml) for various time periods
(4–8 h, lanes 2–7). RNA was extracted and examined by RPA (A).
PhosphorImager quantitation of the blot is shown in (B). Data has
been normalized to GAPDH expression and is represented as a ratio of
VCAM-1/GAPDH mRNA. Representative of three experiments.
induction and/or inhibition were a feature only of
malignant astroglioma cell lines. The proinflammatory
cytokines were able to induce VCAM-1 protein expres-
sion after a 24 h exposure, with TNF-a the strongest
inducer of VCAM-1, followed by IL-1b and IFN-g (Table
2). Induction by all three cytokines was significantly
different from the medium control at the 95% confi-
dence level. In addition, TGF-b1 and TGF-b2 were able
to inhibit the proinflammatory cytokine induction of
VCAM-1 expression by ,33–52%. The 1D.11 and 3C7
antibodies were both able to neutralize the inhibitory
effect of TGF-b2, but only 1D.11 was able to neutralize
the inhibitory effect of TGF-b1 (data not shown).
 TGF-b INHIBITION OF VCAM-1 EXPRESSION 177
TABLE 2. TGF-b inhibits cytokine-induced VCAM-1 expression in
primary human fetal astrocytes

% positive Total %
Cell treatment cells MFIa VCAM-1b inhibition
Medium for 24 h 3.5 6 1.4c 38.1 6 12.1 133.3
TNF-a (10 ng/ml) for
24 h 64.0 6 5.1 158.4 6 47.1 10,137.6
TNF-a 1 TGF-b1
(10 ng/ml) for 24 h 44.7 6 9.4 112.9 6 31.7 5,046.6* 50%d
TNF-a 1 TGF-b2
(10 ng/ml) for 24 h 47.8 6 7.1 102.5 6 28.9 4,899.5* 52%d
IL-1b (1 ng/ml) for
24 h 47.9 6 1.8 89.7 6 15.5 4,296.6
IL-1b 1 TGF-b1 for
24 h 35.4 6 0.9 74.1 6 20.0 2,623.1* 39%e
IL-1b 1 TGF-b2 for
24 h 32.8 6 2.9 68.8 6 18.3 2,256.6* 48%e
IFN-g (100 U/ml) for
24 h 10.0 6 2.3 69.9 6 11.7 699.0
IFN-g 1 TGF-b1 for
24 h 6.5 6 1.8 72.5 6 39.9 471.3* 33%f
IFN-g 1 TGF-b2 for
24 h 6.8 6 0.8 63.1 6 21.1 429.1* 39%f
aMFI 5 Mean Fluorescence Intensity.
bTotalVCAM-1 5 % positive cells 3 MFI.
cMean 6 S.D. of three experiments.
d% inhibition compared to TNF-a alone.
e% inhibition compared to IL-1b alone.
f% inhibition compared to IFN-g alone.

*Statistically different from TNF-a, IL-1b, or IFN-g treatment alone at the 95%
confidence level.

primary human fetal astrocytes by the proinflamma-

Fig. 6. Neutralization of TGF-b1 and TGF-b2 activity with anti-
TGF-b antibodies. U251 cells were seeded at 5 3 105 cells/well in
six-well plates and grown to confluence. Cells were then washed and
incubated in serum-free medium for treatment with TNF-a (10 ng/ml)
plus TGF-b1 (10 ng/ml) (A) or TGF-b2 (10 ng/ml) (B) for 24 h. In
addition, cells were treated with the 1D.11 antibody (3 µg/ml) (which
recognizes all three mammalian forms of TGF-b) or the 3C7 antibody
(3.4 µg/ml) (which recognizes only TGF-b2 and TGF-b3 isoforms),
along with TNF-a plus TGF-b1 or TGF-b2, for 24 h. VCAM-1 protein
expression was assessed by FACS analysis. Total VCAM-1 expression
is calculated as a product of % positive cells 3 mean fluorescence
intensity. Representative of three experiments.
DISCUSSION
This study demonstrates that VCAM-1 protein expres-
sion can be induced on three high grade human astro-
glioma cell lines (U251-MG, D54-MG, CH235-MG) and
tory cytokines TNF-a, IL-1b, and IFN-g, with TNF-a
the strongest inducer of VCAM-1. TGF-b (both b1 and
b2) was able to inhibit cytokine-induced VCAM-1 ex-
pression in these cells as well. Optimal inhibition of
cytokine-induced VCAM-1 expression was observed
when TGF-b was added simultaneously with the induc-
ing stimuli, and preincubation with TGF-b actually
diminished the extent of inhibition. The inhibitory
effect of TGF-b was abrogated by the inclusion of
neutralizing antibodies against TGF-b1 and TGF-b2,
demonstrating the specificity of the response. In addi-
tion, cytokine-induced VCAM-1 mRNA expression was
significantly inhibited in the presence of TGF-b.
Our results indicate that both TGF-b1 and TGF-b2
can inhibit VCAM-1 expression (mRNA and protein)
induced by three proinflammatory cytokines, TNF-a,
IL-1b, and IFN-g. The inhibitory effect of TGF-b was
mediated in a dose-dependent fashion, and the extent of
inhibition was most pronounced when TGF-b was added
simultaneously with the proinflammatory cytokines.
There have been few studies on the ability of TGF-b to
inhibit VCAM-1 expression; Gable et al. (1995) have
shown that TGF-b inhibits TNF-a induced VCAM-1
expression in smooth muscle cells, similar to what we
have observed in the astroglioma cell lines and primary
human astrocytes. We also documented that although
IFN-g induction of VCAM-1 expression in astroglioma
lines and human astrocytes is low compared to TNF-a/
IL-1b induction, TGF-b is capable of inhibiting IFN-g
induced VCAM-1 expression. These findings are of
interest since we have shown that ICAM-1 expression
in astroglioma cell lines and primary astrocytes is
differentially regulated by TGF-b (Shrikant et al.,
1996). In those studies, TNF-a and IL-1b induced
178 WINKLER AND BENVENISTE
ICAM-1 expression was inhibited by TGF-b, while IFN-g
induced ICAM-1 expression was unaffected, demonstrat-
ing that TGF-b acts in a stimulus-specific manner to
inhibit ICAM-1 expression (Shrikant et al., 1996). In
addition, there are differences in the kinetics by which
TGF-b can inhibit gene expression in glial cells. In this
study, TGF-b exerted its most potent inhibitory effect
when added simultaneously with the inducing cytokines,
and preincubation with TGF-b did not enhance the
inhibitory effect; in fact, the extent of inhibition slightly
diminished with TGF-b pretreatment. In contrast,
TGF-b inhibition of TNF-a and IL-1b induced ICAM-1
expression required a pretreatment time of ,12 h for
the inhibitory effect to be observed, and simultaneous
addition of TGF-b did not inhibit ICAM-1 gene expres-
sion (Shrikant et al., 1996). Thus there are clearly
differences in the ability of TGF-b to regulate ICAM-1
and VCAM-1 expression in cells of astrocytic origin.
The mechanism by which TGF-b inhibits VCAM-1
expression is not understood. TGF-b may downregulate
the receptors for the inducing stimuli or affect the
ability of the receptors to bind ligand (Dubois et al.,
1990). TGF-b has been shown to induce expression of
the IL-1 receptor antagonist (Wahl et al., 1993) which
could explain the TGF-b inhibitory effect on IL-1b
induced VCAM-1. Additionally, TGF-b may inhibit
VCAM-1 gene expression by affecting the transcription
factors involved in this process. Cytokine-mediated
transcriptional activation of VCAM-1 requires two
tandem binding sites for NF-kB located in the VCAM-1
promoter at positions of 273 and 258, as well as the
binding site for interferon regulatory factor-1 (IRF-1)
(Collins et al., 1995; Neish et al., 1995). In this regard,
TGF-b has been shown to inhibit NF-kB activity in
B-cells by increasing the expression of IkBa, a specific
inhibitor of NF-kB (Arsura et al., 1996). Future studies
will focus on the molecular mechanisms underlying the
inhibitory effect of TGF-b on VCAM-1 expression.
Clearly the regulation of cytokine-induced adhesion
molecule expression is a complex matter. Our results
were obtained using transformed cell lines and fetal
astrocytes, thus in vivo regulation of VCAM-1 expres-
sion in the adult diseased brain may differ from our in
vitro findings. Nonetheless, pharmacologic and immu-
nologic inhibition of leukocyte-astrocyte adhesion at
the BBB represents a focus for research directed at
attenuating inflammatory tissue injury by controlling
leukocyte trafficking into the CNS. The identification of
the regulatory effects of TGF-b on VCAM-1 expression
on astrocytes suggests the potential for pharmacologic
approaches to diseases such as MS and AIDS dementia
complex with a disrupted BBB. Further understanding
of the mechanisms involved in cytokine-induced adhe-
sion molecule suppression by TGF-b will be necessary
to consider this type of immunotherapy for a diverse
spectrum of CNS diseases.
ACKNOWLEDGMENTS
The authors thank Dr. Bruce Pratt (Genzyme Corpo-
ration, Cambridge, MA) for the 1D.11 and 3C7 antibod-
ies and TGF-b2, Dr. Eugene Major (National Institutes
of Health, Bethesda, MD) for the human fetal astro-
cytes, and Sue Wade for excellent secretarial and
editorial assistance.
This work was supported in part by the National
Multiple Sclerosis Society (RG-2269) and the National
Institutes of Health [NS29719, NS34856 and MH55795
(E.N.B.)]. M.K.W. is the recipient of the Dixon Postdoc-
toral Fellowship Grant, which also supports this work.
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