Download Enhanced B7-2 Gene Expression by Interferon

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PHAGOCYTES
Enhanced B7-2 Gene Expression by Interferon-␥ in Human Monocytic Cells Is
Controlled Through Transcriptional and Posttranscriptional Mechanisms
By R.E. Curiel, C.S. Garcia, S. Rottschafer, M.C. Bosco, and I. Espinoza-Delgado
B7-2 is a costimulatory molecule expressed on professional
antigen-presenting cells that provides T cells with a critical
signal resulting in T-cell activation. Interferon-␥ (IFN-␥) enhances B7-2 protein expression in monocytic cells. However,
the molecular mechanisms controlling the enhanced expression of B7-2 are poorly understood. Northern blot and flow
cytometry analysis revealed that human monocytes and the
human monocytic cell line MonoMac6 (MM6) constitutively
expressed B7-2 mRNA and protein and IFN-␥ treatment
further enhanced the expression of both molecules. The
ability of IFN-␥ to enhance B7-2 mRNA was evident at the
dose of 31 U/mL and reached plateau levels at 500 U/mL. The
effects of IFN-␥ on B7-2 mRNA expression were time depen-
dent and occurred within 3 hours of treatment and increased
through 24 hours. In vitro transcription assays and mRNA
stability experiments showed that IFN-␥ increases both
transcriptional activity and the stability of B7-2 mRNA.
Treatment of MM6 cells with cycloheximide showed that de
novo protein synthesis was not required for the IFN-␥–
enhanced expression of B7-2 mRNA. Overall, these studies
show for the first time that IFN-␥–enhanced expression of
B7-2 protein in human monocytic cells is controlled at the
gene level through a dual mechanism involving transcriptional and posttranscriptional mechanisms.
r 1999 by The American Society of Hematology.
T
costimulatory signal.10,13,14 B7-1 knockout (KO) mice display
relatively normal Th1- and Th2-dependent immune responses,
whereas B7-2 KO mice are severely immunocompromised.11
On most resting professional APCs, B7-2, but not B7-1, is
constitutively expressed. Noteworthy, on activation of dendritic
cells (DC), macrophages, and B cells, B7-2 expression can be
induced with faster kinetics and, in general, to a much higher
level of expression than B7-1.15 Although the particular dominance of B7-1 or B7-2 as the main costimulatory molecule in
vitro or in vivo is yet to be clearly determined, collectively these
findings indicate that B7-2 is a major player in T-cell costimulation, and it has a critical dominant role in the initiation of the
immune response.
Interferon-␥ (IFN-␥), secreted by activated T lymphocytes
and natural killer cells,16 is a potent activator of human
monocytic cells. IFN-␥ activates and differentiates human
monocytes,17,18 leading to increase in Class I and Class II MHC
expression,16,19,20 transferrin receptor expression,21 toxic oxygen derivatives and nitric oxide production,22 tumoricidal
activity,17,23 and regulation of cytokine expression24-27 and their
receptors.27 Recently, it has been shown that IFN-␥ also
increases the expression of the costimulatory molecule B7-2 in
both human monocytes28 and murine macrophages.13 However,
the molecular mechanisms controlling the expression of B7-2 in
human monocytic cells are not well understood. Elucidating the
mechanisms involved in the expression of this important
costimulatory molecule is essential for the understanding of the
regulation of T-cell–dependent immunity. To investigate the
mechanisms involved in B7-2–enhanced expression by IFN-␥,
we studied the regulation of B7-2 mRNA in the human
monocytic cell line MonoMac6 (MM6). MM6 cells have been
extensively characterized, and they display phenotypic and
functional features of mature human monocytes,29 including
enhanced antigen expression in response to IFN-␥. We show
that IFN-␥ enhances B7-2 mRNA accumulation and protein
expression in human monocytic cells. Upregulation of B7-2
expression by IFN-␥ occurs rapidly and through mechanisms
that do not require new protein synthesis. Finally, we also
provide first evidence that the enhancement of B7-2 mRNA
expression by IFN-␥ is controlled through both transcriptional
and posttranscriptional mechanisms.
HE ENGAGEMENT of the antigen-specific T-cell receptor
(TCR) with an antigenic peptide in the context of the
major histocompatibility complex (MHC) provides the initial
signal, recognition, for a T-cell–mediated antigen-specific immune response.1 However, although signaling through the TCR
is essential for proper activation of T cells, a second signal
termed costimulation seems to be pivotal in controlling the
functional outcome of T-cell activation.1-3 Absence of a costimulatory signal can induce long-term antigen-specific T-cell unresponsiveness called anergy.1-3 T-cell costimulation occurs after
interaction of the CD28 T-cell receptor with one of the B7
family members expressed on an antigen-presenting cell (APC).
The B7 family of proteins B7-1 and B7-2 are both members of
the Ig superfamily, and their functional status displays a
restricted APC-dependent pattern of expression.4,5 Several
transfection studies have indicated that both B7-1 and B7-2 can
serve as ligands for in vitro costimulation of T cells.6-10
Although either of the B7 family members can provide costimulation, mounting evidence has accumulated supporting a dominant role for B7-2 in primary T-cell responses.11 Recent
observations have indicated that anti–B7-1 antibodies have a
minimal inhibitory effect on T-cell proliferation during allogeneic primary mixed lymphocyte reactions,12 whereas in most
cases, treatment with anti–B7-2 antibodies inhibits most of the
early T-cell activation responses to levels similar to those
observed with hCTLA4Ig, a potent inhibitor of the B7/CD28
From Department of Medicine and Stanley S. Scott Cancer Center,
Louisiana State University Medical Center, New Orleans, LA; and
Laboratorio di Biologia Molecolare, Instituto Giannina Gaslini, Genova
Quarto, Italy.
Submitted September 30, 1998; accepted April 26, 1999.
Address reprint requests to I. Espinoza-Delgado, MD, 1542 Tulane
Ave, Hematology-Oncology Suite 604K, New Orleans, LA 70112;
e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1999 by The American Society of Hematology.
0006-4971/99/9405-0004$3.00/0
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Blood, Vol 94, No 5 (September 1), 1999: pp 1782-1789
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MOLECULAR REGULATION OF B7-2
MATERIALS AND METHODS
MM6 cell culture and cytokine stimulation. The human monocytic
cell line MM629 was obtained from the repository of the National
Cancer Institute, Frederick Cancer Research and Development Center
(Frederick, MD) and cultured as previously described.29 Briefly, MM6
cells were incubated at 37°C in a 5% CO2-humidified atmosphere in
Dulbecco’s modified Eagle’s medium (DMEM) tissue culture (Advance
Biotechnology, Columbia, MD) supplemented with 50 U/mL penicillin,
50 µg/mL streptomycin, 2 mmol/L L-glutamine, and 15% heatinactivated fetal bovine serum (FBS) (Hyclone Laboratories, Logan,
UT). Exponentially growing cells were cultured at 5 ⫻ 105 cells/mL in
complete medium, hereafter referred to as complete medium. Ten
milliliters of cell suspension were plated in 100-mm2 tissue culture
plates (Corning Glass Works, Corning, NY) in medium alone or
supplemented with the indicated doses of recombinant human IFN-␥
(specific activity 2.02 ⫻ 107 U/mg), kindly provided by Dr Craig W.
Reynolds (Biological Resources Branch, Division of Cancer Treatment,
Diagnosis and Centers, National Cancer Institute, Frederick Cancer
Research and Development Center). Cells were harvested at the
indicated time points and were used for flow cytometry analysis or total
RNA extraction as described below.
Monocyte isolation, culture condition, and stimulation. Peripheral
blood leukocytes were obtained from normal healthy volunteers by
leukapheresis using a Fenwell CS-3000 blood cell separator (Fenwell
Laboratories, Deerfield, IL). Mononuclear cells were separated by
density gradient centrifugation on lymphocyte separation medium
(LSM; Organum Teknika Corp, Durham, NC) and then purified in
suspension from the unfractionated mononuclear leukocyte preparation
by counter-current centrifugal elutriation in a Beckman JE-6 elutriation
chamber and rotor system (Beckman Instruments Inc, Palo Alto, CA),
as described elsewhere.27 The purity of monocyte preparations was
94% ⫾ 3%, as assessed by morphology on Giemsa-stained cytocentrifuge slide preparations and by flow cytometry using the monocytespecific monoclonal antibody (MoAb) Leu M3 (Becton Dickinson,
Mountain View, CA). Viability, as determined by trypan blue exclusion
test, was ⬎99%. Monocytes were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD), supplemented with 100 U/mL penicillin, 100
U/mL streptomycin, 2 mmol/L L-glutamine, 20 mmol/L HEPES
(GIBCO-BRL, Gaithersburg, MD), and 10% heat-inactivated FBS
(Hyclone Laboratories). Monocytes were cultured at the indicated time
point in 15-cm Lux plates (Miles Scientific, Wapersville, CA) at 2 ⫻ 106
cells/mL in medium alone or supplemented with either 500 U/mL of
recombinant human IFN-␥ (specific activity 2.02 ⫻ 107 U/mg) or 10
ng/mL of lipopolysaccharide (LPS) from E coli serotype 0111:B4
purchased from Sigma Chemical Company (St Louis, MO).
Flow cytometry analysis. Flow cytometry analysis was performed
as previously described.30 Briefly, monocytic cells were washed once
with phosphate-buffered saline (PBS) and then labeled as described
below. MM6 cells (1 ⫻ 106) were resuspended in 100 µL of PBS
containing 2% heat-inactivated human AB serum (Sigma Chemical Co)
and 0.05% sodium azide (Sigma Chemical Co), hereafter referred to as
flow cytometry buffer (FB). Cells were then incubated for 30 minutes at
4°C with anti-CD86 (B70/B7-2; Pharmingen, San Diego, CA) or with
isotype control anti-␥1/␥2a (Becton Dickinson, San Jose, CA) antibodies, and concentrations were used according to manufacturer’s recommendations. Cells were washed twice with cold FB and fixed with FB
buffer containing 0.5% paraformaldehyde. Flow cytometry analysis of
B7-2 expression was performed using an Elite flow cytometer (Coulter
Corp, Hialeah, FL). The data are expressed as mean channel fluorescence intensity (MCFI in arbitrary units of fluorescence). MCFI is an
indication of the relative density of surface antigens present on
individual cells.
Northern blot analysis. Human peripheral blood monocytes or
MM6 cells were cultured in medium alone or supplemented with the
1783
indicated reagents. Total RNA was extracted by lysis with TRIzol
(GIBCO-BRL, Gaithersburg, MD) and purified according to the
manufacturer’s specifications. Northern blot analysis was performed in
accordance with the previously described protocol.30 Twenty micrograms of total RNA from each sample was electrophoresed under
denaturing conditions, blotted onto nytran membranes (Schleicher &
Schuell Inc, Keene, NH), and cross-linked by UV irradiation. Membranes were prehybridized at 42°C in Hybrisol (Oncor Inc, Gaithersburg, MD) and hybridized overnight with 2 ⫻ 106 cpm/mL of
32P-labeled probe. Membranes were then washed three times at room
temperature for 10 minutes in 2⫻ SSC (1⫻ SSC ⫽ 0.15 mol/L NaCl,
0.015 mol/L sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate
(SDS), and twice at 65°C for 20 minutes in 0.2⫻ SSC and 0.1% SDS
before being autoradiographed using Kodak Biomax-MR (Eastman
Kodak Company, Rochester, NY) films and intensifying screens at
⫺70°C. The human cDNA B7-2 probe (a kind gift from Dr Gordon
Freeman, Division of Hematologic Malignancies, Dana-Farber Cancer
Institute, and Department of Medicine, Harvard Medical School,
Boston, MA) and human glyceraldehide-3 phosphate dehydrogenase
(GAPDH) (Clontech, Palo Alto, CA) were labeled by random priming
and [␣-32P]dCTP (3,000 Ci/mmol; Amersham, Arlington Heights, IL).
For mRNA synthesis inhibition, actinomycin D (Act-D; Sigma Chemical Co) was dissolved in ethanol at 1 mg/mL and used at a final
concentration of 5 µg/mL, as indicated in the text. For protein synthesis
inhibition experiments, cycloheximide (CHX; Sigma Chemical Co) was
used at a final concentration of 10 µg/mL. An alphaImager 2000 (Alpha
Innotech Corporation, San Leandro, CA) was used to analyze the bands’
intensities of the autoradiographs of the Northern blots. The graphs
were generated from the average of the intensities of the three distinct
species of B7-2 mRNA. The results were normalized to GAPDH.
Nuclear run-on. Nuclear run-on experiments were performed as
previously described.18 Briefly, nuclei were isolated from 5 ⫻ 107
cells/sample by lysing cells in 4 mL of lysis buffer (10 mmol/L Tris-HCl
pH 7.4, 3 mmol/L MgCl2, 10 mmol/L NaCl, 150 mmol/L sucrose, and
0.5% nonidet P-40; Sigma Chemical Co) for 5 minutes on ice. Nuclei
were spun at 167g for 5 minutes at 4°C, and pellets were resuspended in
lysis buffer without nonidet P-40. Nuclei were pelleted again as
described above and resuspended in 150 µL of freezing buffer (50
mmol/L Tris-HCl pH 8.3, 40% glycerol, 5 mmol/L MgCl2, 0.1 mmol/L
EDTA). Run-on assays were performed by adding 150 µL of 2⫻
transcription buffer (20 mmol Tris-HCl pH 8.0, 300 mmol KCl, 10
mmol/L MgCl2, 200 mmol/L sucrose 20% glycerol, 1 mmol dithiotreitol,
0.5 mmol each of adenosine triphosphate [ATP], guanosine triphosphate
[GTP], and cytidine triphosphate [CTP]) and 100 µCi of 800 Ci/mmol
[␣32P] uridine triphosphate (NEN, Boston, MA) to 150 µL of nuclei
suspension. The samples were incubated at 29°C for 30 minutes. Thirty
microliters of 200 mmol CaCl2 and 30 µL of 1 U/mL Rnase-free Dnase
1 (Promega, Gaithersburg, MD) were added to each reaction and further
incubated for 10 minutes at 29°C. Labeled transcripts were isolated
using TRIzol (GIBCO-BRL) and purified according to the manufacturer’s specifications. Equal amounts of radioactivity (about 2 ⫻ 106 cpm
of labeled RNA) were added in 2 mL of Hybrizol (Oncor Inc) to nytran
membranes on which 500 ng of denatured full-length human B7-2
cDNA (1.1 kb) and chicken ␤-actin cDNA (1.8 kb, HindIII fragment;
Oncor Inc) were immobilized using a slot blot apparatus (GIBCO-BRL)
and a UV crosslinker (Fisher Scientific, Pittsburgh, PA). Hybridization
was performed at 42°C for 48 hours. Filters were washed three times at
42°C for 15 minutes with 2⫻ SSC/0.1% SDS and two times at 65°C for
20 minutes with 0.2⫻ SSC/0.1% SDS. Filters were then autoradiographed at ⫺70°C. Data were normalized for the content of ␤-actin
present in each sample using an alphaImager 2000 (Alpha Innotech
Corporation).
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1784
CURIEL ET AL
RESULTS
IFN-␥ enhances B7-2 mRNA expression in MM6. To determine whether MM6 cells responded to IFN-␥ with changes in
B7-2 mRNA expression, MM6 were cultured for 18 hours in
medium alone or in the presence of 500 U/mL of IFN-␥, a dose
previously shown to induce maximal activation of human
monocytes.23 Total RNA was extracted, and Northern blot
analysis was performed. As shown in Fig 1, a low basal
expression of B7-2 mRNA was detected in the medium control,
whereas IFN-␥ treatment of MM6 cells led to a significant
enhanced expression of B7-2 mRNA. Dose-response experiments were performed to determine the optimal concentration
of IFN-␥ needed to induce maximal enhanced expression of
B7-2 mRNA. MM6 cells were cultured in medium alone or in
the presence of increasing concentrations of IFN-␥. After 18
hours of stimulation, total RNA was extracted and analyzed by
Northern blot for B7-2 mRNA expression. As seen in Fig 2,
IFN-␥ induced a dose-dependent increase of B7-2 mRNA. As
little as 31 U/mL were sufficient to induce a modest increase in
B7-2 mRNA levels, whereas doses of IFN-␥ between 250 and
1,000 U/mL were required for maximal expression. Therefore,
500 U/mL of IFN-␥ was used in all subsequent experiments.
To determine the kinetics of upregulation of B7-2 mRNA by
IFN-␥, MM6 cells were incubated in medium alone or with 500
U/mL of IFN-␥ for the indicated lengths of time. Total RNA was
extracted, and a Northern blot analysis was performed to detect
B7-2 mRNA. As seen in Fig 3, an early enhanced expression of
Fig 2. B7-2 mRNA expression is enhanced in a dose-dependent
manner by IFN-␥. MM6 cells were cultured for 18 hours in the absence
or presence of increasing concentrations of IFN-␥. Total cellular RNA
was extracted and analyzed by Northern blot for B7-2 mRNA expression. The same membrane was rehybridized with GAPDH to control
that equal amounts of RNA were loaded in each lane. Data shown are
from 1 representative experiment of 2 performed. Northern blot
analysis for B7-2 mRNA expression (upper panel). Quantitative analysis of B7-2 mRNA expression (lower panel). As described in Materials
and Methods, the bands’ intensities were normalized to the GAPDH
housekeeping gene control, and the graph was generated with the
relative values obtained after normalization.
Fig 1. IFN-␥ treatment of MM6 cells increases B7-2 mRNA expression. MM6 cells were cultured for 18 hours in the absence or presence
of 500 U/mL of IFN-␥. Total cellular RNA was extracted and analyzed
by Northern blot for B7-2 mRNA expression. The same membrane
was rehybridized with GAPDH to control that equal amounts of RNA
were loaded in each lane. Data shown are from 1 representative
experiment of 4 performed.
the B7-2 mRNA was observed within 3 hours after stimulation
with IFN-␥. Steady increases in B7-2 mRNA expression were
noticed from 6 hours (a twofold increase) to 24 hours (a
sevenfold increase) above the levels seen in medium-treated
MM6 cells.
To evaluate whether the enhanced expression of B7-2 mRNA
led to surface expression of B7-2, MM6 cells were cultured in
the absence or presence of 500 U/mL of IFN-␥ and analyzed by
flow cytometry at 24 hours. As seen in Fig 4, medium-treated
monocytic cells constitutively expressed low basal levels of
B7-2 surface protein. Upon IFN-␥ treatment, a threefold
increase of B7-2 expression was observed over the MCFI of the
control groups by 24 hours. Similar results were obtained in
three different experiments with the IFN-␥–enhanced B7-2
expression, ranging from twofold to threefold. For any given
experiment, between 50% to 70% of MM6 cells were positive
for B7-2 surface expression, but no significant differences in the
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MOLECULAR REGULATION OF B7-2
1785
but reproducible increase in transcriptional activity was observed over the basal level of transcription of the mediumtreated cells (data not shown).
IFN-␥ enhances B7-2 mRNA stability. Experiments were
performed to determine whether IFN-␥ influenced the stability
of B7-2 mRNA. MM6 cells were incubated for 18 hours with
medium alone or supplemented with 500 U/mL of IFN-␥. After
the 18-hour incubation period, Act-D was added to the cultures
for the indicated lengths of time to block further RNA transcription. Northern blot analysis revealed that B7-2 mRNA decayed
with different kinetics in untreated and IFN-␥–treated cells (Fig
6). The level of B7-2 mRNA in medium-treated cells decreased
by 50% (T1⁄2) after 2 hours and 20 minutes, and B7-2 mRNA
became almost undetectable after 4 hours of Act-D treatment.
On the other hand, IFN-␥–treated cells displayed an enhanced
B7-2 mRNA stability resulting on a T1⁄2 of 4 hours. Similar
results were observed in two independent experiments. Taken
together, these results showed that IFN-␥ enhancement of B7-2
gene expression in MM6 cells occurs through a dual mechanism
involving transcriptional and posttranscriptional levels of regulation.
Protein synthesis is not required for the IFN-␥–induced
upregulation of B7-2 mRNA. To determine whether active
protein synthesis was necessary for the IFN-␥ upregulation of
B7-2 mRNA, MM6 cells were incubated for 12 hours in the
absence or presence of 500 U/mL of IFN-␥ and in the absence
or presence of the protein-synthesis inhibitor CHX. As shown in
Fig 7, the addition of CHX to IFN-␥–treated MM6 cells did not
Fig 3. Kinetics of IFN-␥–induced upregulation of B7-2 mRNA. MM6
cells were stimulated in the presence or absence of 500 U/mL of IFN-␥
for the indicated times. Total cellular RNA was isolated, and Northern
blot analysis for B7-2 mRNA expression was performed. The same
filter was subsequently probed with GAPDH to ensure that comparable amounts of RNA were loaded in each lane. Data shown are from
1 representative experiment of 2 performed. Northern blot analysis
for B7-2 mRNA expression (upper panel). Quantitative analysis of
B7-2 mRNA expression (lower panel). As explained in Materials and
Methods, the bands’ intensities were normalized to the GAPDH
housekeeping gene control, and the graph was generated with the
relative values obtained after normalization.
percentage of positive cells were observed between the IFN-␥–
treated cells and the medium-treated controls (data not shown).
These results indicate that IFN-␥–enhanced B7-2 mRNA
expression in MM6 cells is associated with an increased
expression of B7-2 protein on the surface of these cells.
The transcriptional activity of the B7-2 gene is enhanced by
treatment with IFN-␥. To investigate whether the increased
expression of B7-2 by IFN-␥ involved changes in B7-2 gene
transcription, nuclear run-on experiments were performed.
MM6 cells were incubated with medium alone or supplemented
with 500 U/mL of IFN-␥. The nuclei were isolated at 2 hours
and 4 hours after treatment, and nuclear run-on assays were
performed. As seen in Fig 5, B7-2 gene was transcriptionally
active in medium control cells. A twofold increase in the rate of
transcription of the B7-2 gene was observed 4 hours after IFN-␥
treatment. As early as 2 hours post–IFN-␥ treatment, a moderate
Fig 4. B7-2 surface expression is enhanced by treatment with
IFN-␥. MM6 cells were cultured for 24 hours in the presence or
absence of 500 U/mL of IFN-␥. Cells were harvested and labeled for
immunofluorescence analysis as described in Materials and Methods.
Flow cytometry analysis was performed using an Elite flow cytometer. Closed histogram (in black) represents isotype-matched antibody control. Broken line histogram (-----) represents medium treated
cells (MCFI ⴝ 16). Solid line histogram (_____) represents IFN-␥–
treated cells (MCFI ⴝ 48). The MCFI values shown for medium- and
IFN-␥–treated cells indicate the relative MCFI of the B7-2 staining
monoclonal antibody after subtracting the MCFI of the isotypematched antibody. Data shown are from 1 of 3 similar experiments.
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1786
CURIEL ET AL
performed. As shown in Fig 8, a basal expression of B7-2
mRNA was detected in the medium-treated cells. IFN-␥–treated
human monocytes displayed a major increase of B7-2 mRNA
expression that led to an enhanced B7-2 surface expression
(data not shown). On the other hand, the powerful monocyte
activator LPS did not affect the basal level expression of B7-2
mRNA. These results remained unchanged up to 14 hours (data
not shown).
DISCUSSION
Recent studies have shown that IFN-␥ increases the expression of B7-2 in a number of different APCs,13,28,31-33 yet the
mechanisms involved in this induction remain poorly understood. The present study provides the first look at the molecular
mechanisms involved in the IFN-␥–enhanced expression of the
B7-2 gene in human monocytic cells. The human monocytic
cell line MM6 was used to study the effects of IFN-␥ on B7-2
gene expression, given that these cells are phenotypically and
functionally similar to mature human monocytes.29 Our data
show that as little as 31 U/mL of IFN-␥ was sufficient to induce
an increase of B7-2 transcripts. B7-2 mRNA–enhanced accumulation was followed by an increased level of B7-2 surface
expression, suggesting that IFN-␥–driven B7-2–enhanced surface expression is controlled, at least in part, at the gene level.
To investigate whether the changes induced by IFN-␥ in the
MM6 monocytic cell line were also observed in primary cells,
we performed Northern blot analysis in human monocytes. Our
data showed that IFN-␥ also rapidly upregulated the expression
of B7-2 mRNA in human monocytes. Furthermore, the upregulated expression of B7-2 by IFN-␥ was stimulus-specific and
not associated with a general monocyte-activated phenotype,
because a well-known monocyte activator LPS failed to upregulate B7-2 mRNA expression. IFN-␥–enhanced B7-2 expression
Fig 5. IFN-␥ augments B7-2 gene transcription. MM6 cells (5 ⫻ 107
cells/point) were treated with medium alone or with 500 U/mL of
IFN-␥. Nuclei were isolated at the indicated time points, and the rate
of transcription of the B7-2 gene was then assessed by nuclear run-on
analysis as described in Materials and Methods. Data presented are
from 1 of 2 similar experiments. The graph was generated as
described in Materials and Methods, with the relative values obtained
after normalization of the bands’ intensities to the respective amounts
of ␤-actin.
decrease the enhanced B7-2 mRNA expression. Noteworthy,
addition of CHX to medium-treated MM6 cells caused a
superinduction of the basal B7-2 mRNA expression. These
results suggest that the IFN-␥–induced upregulation of B7-2
mRNA expression is not dependent on de novo protein synthesis.
IFN-␥ enhances B7-2 mRNA expression in human peripheral
blood monocytes. To determine whether the IFN-␥–induced
changes in B7-2 mRNA expression seen in MM6 cells were also
present in primary human monocytes, peripheral blood monocytes were cultured for 3 hours in medium alone or in the
presence of either 500 U/mL of IFN-␥ or 10 ng/mL of LPS.
Total RNA was extracted, and Northern blot analysis was
Fig 6. Treatment of MM6 cells with IFN-␥ increases B7-2 mRNA
stability. MM6 cells were incubated for 18 hours in medium alone or
in medium supplemented with 500 U/mL of IFN-␥. After 18 hours,
cells were treated with 5 ␮g/mL of Act-D, and their total cellular RNA
was collected and analyzed by Northern blot for B7-2 mRNA expression at the indicated time points. Data shown are from 1 representative experiment of 2 performed. The graph was generated as described in Materials and Methods, and data are presented as the
relative amounts of B7-2 mRNA remaining after adding Act-D and
normalizing to the respective amounts of GAPDH.
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MOLECULAR REGULATION OF B7-2
1787
contribute to the increased accumulation of B7-2 mRNA in
MM6 cells. Rapid degradation of mRNAs encoding many
oncogenes and cytokines are regulated in part by A⫹U-rich
elements (AREs) in their 38-untranslated regions (38UTR).34,35
Proteins that bind to AREs in the 38UTR of these messages
control their stability, and by doing so, they also control the
levels and timing of expression.34,35 Computer analysis of the
published B7-2 mRNA sequence9 (accession number L25259)
failed to reveal any AREs in the 38UTR of B7-2 gene,
suggesting that the increased stability of B7-2 mRNA observed
in MM6 cells after IFN-␥ treatment is not regulated or
controlled by AREs. These results suggest that novel regulatory
elements or mechanisms other than A⫹U-rich regions are
responsible for the enhanced T1⁄2 of the B7-2 mRNA observed
in IFN-␥–treated MM6 cells. Interestingly, a novel protein
complex has recently been reported that binds to the 38UTR of
tumor necrosis factor-␣ (TNF-␣) mRNA independently of the
presence of AREs.36,37 This report and our present observation
clearly suggest that other, yet to be identified, regulatory
element(s) can control the stability of the mRNA. We are
currently undertaking efforts to determine whether IFN-␥
affects the protein binding pattern of the 38UTR of B7-2
mRNA.
To further dissect the mechanism responsible for the IFN-␥–
enhanced B7-2 mRNA expression, nuclear run-on experiments
were performed. These in vitro transcription assays showed that
B7-2 gene was transcriptionally active in medium-treated MM6
cells and that IFN-␥ treatment further increased B7-2 gene rate
of transcription. Our results provide the first evidence that
IFN-␥ can exert transcriptional control on this gene and that this
Fig 7. De novo protein synthesis is not required for the IFN-␥–
induced enhance expression of B7-2 mRNA. MM6 cells were incubated for 12 hours in the absence or presence of 500 U/mL of IFN-␥
and in the absence or presence of 10 ␮g/mL of CHX. Total cellular
RNA was extracted and analyzed by Northern blot for B7-2 mRNA
expression (upper panel). Data shown are from 1 of 3 similar
experiments. The graph represents the quantitative analysis of B7-2
mRNA expression (lower panel). As stated in Materials and Methods,
the bands’ intensities were normalized to the GAPDH housekeeping
gene control, and the graph was generated with the relative values
obtained after normalization.
was noticed as early as 3 hours and further augmented thereafter
until 24 hours. The rapid upregulation of B7-2 mRNA in MM6
cells and primary cultured monocytes suggests a direct effect of
IFN-␥ on the expression of this gene rather than a secondary
effect mediated by an IFN-␥ inducible gene.
In an attempt to determine whether message stabilization was
one of the mechanisms responsible for the enhanced B7-2 gene
expression by IFN-␥, the T1⁄2 of the B7-2 mRNA was studied.
After blocking new RNA synthesis with Act-D, B7-2 mRNA
decayed at a slower rate in IFN-␥–treated MM6 cells than in
medium-treated cells. These results clearly indicate that IFN-␥
increased the T1⁄2 of the B7-2 transcripts and that this effect may
Fig 8. IFN-␥ treatment of human peripheral blood monocytes
augments B7-2 mRNA expression. Human peripheral blood monocytes were cultured for 3 hours in medium alone or in the presence of
either 500 U/mL of IFN-␥ or 10 ng/mL of LPS. Total cellular RNA was
extracted and analyzed by Northern blot for B7-2 mRNA expression.
The same membrane was rehybridized with GAPDH to control that
equal amounts of RNA were loaded in each lane. Data shown are from
1 representative experiment of 2 performed.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1788
effect contributes, at least in part, to the enhanced expression of
B7-2 message seen in MM6 after IFN-␥ stimulation.
The early IFN-␥–enhanced B7-2 mRNA expression in MM6
cells suggested a direct response independent of de novo protein
synthesis. Inhibition, as well as superinduction of gene expression, have been reported in monocytes treated with different
stimuli in the presence of the protein synthesis inhibitor
CHX.38,39 Our data showed that treatment with CHX superinduced B7-2 mRNA basal expression. These results indicate that
no new protein synthesis is required for the constitutive or
IFN-␥–enhanced expression of B7-2 mRNA and suggest that
B7-2 expression may be controlled by a de novo synthesized
repressor protein(s) and/or by a factor(s) involved in regulation
of mRNA stability. These two mechanisms have been previously suggested to be operating in the superinducibility of
cytokine genes by CHX.39-41
The group of genes that are under direct transcriptional
control of IFN-␥ and that do not require the synthesis of new
transcriptional factors for their expression are referred to as
primary response genes.42-45 Some of the primary response
genes are transcriptional factors themselves, such as interferon
regulatory factor 1 (IRF-1), interferon regulatory factor 2
(IRF-2), MHC class II transactivator (CIITA), interferon consensus sequence binding protein (ICSBP), p48, ␥-responsive factor
1 (␥RF-1), and signal transducer and activator of transcription
(STAT1␣), which in turn regulate the expression of so-called
secondary IFN-␥ response genes.16 The rapid upregulation of
B7-2 gene transcriptional activity by IFN-␥, independent of de
novo protein synthesis, suggests a direct effect on the expression of this gene similar to that observed in primary response
genes such as guanylate-binding protein (GBP), monokine
induced by gamma interferon (mig), Fc␥RI, peptide transporter
1 (TAP1), and others.16 Based on the published observations by
others and our current results, it is tempting to speculate that
B7-2 might be an IFN-␥ primary response gene, and if so, its
promoter should contain IFN-␥ responsive elements. Although
recently the 58 untranslated region of the murine B7-2 gene was
partially characterized, the human and murine B7-2 promoters
remain elusive. We are currently undertaking efforts in our
laboratory to further characterize the 58 untranslated region of
the human B7-2 gene.
The data presented here provides the first report dissecting
the molecular mechanisms involved in the IFN-␥–induced B7-2
gene upregulation. We clearly showed that through processes
not requiring new protein synthesis, a dual mechanism involving transcriptional and posttranscriptional changes is responsible for the enhanced expression of B7-2 mRNA in IFN-␥–
treated MM6 cells. This complex and tight regulation of B7-2
gene expression underscores its relevance in the immune
response and may provide monocytic cells with the capacity to
quickly upregulate the expression of B7-2.
One of the novel therapeutic approaches that is currently
being explored against malignancies is the manipulation of
costimulatory molecules expressed on APCs or on tumor cells
engineered to act as APCs in the context of cancer vaccines.
Costimulation of T cells by B7-2 may play a critical role in
determining the outcome of the immune response that may
range from immunity to tolerance. Therefore, understanding the
mechanisms regulating B7-2 expression is particularly impor-
CURIEL ET AL
tant because it may provide a rational basis for the development
of novel anticancer therapeutic modalities. The complex nature
of the B7-2 gene regulation and its pivotal role in T-cell
costimulation warrant further investigation of the transcriptional and posttranscriptional events controlling its expression.
ACKNOWLEDGMENT
The authors thank Dr Paul L. Fidel, Jr, Dr Ronald B. Luftig, and Dr
James M. Mwatibo for their critical review of this manuscript. They also
thank Dr Gordon Freeman for kindly providing the human B7-2 cDNA,
Cynthia G. Healy for generating the flow cytometry histogram figure for
this paper, and Marilyn Schoen, RN, for performing cytapheresis.
REFERENCES
1. Jenkins MK, Ashwell JD, Schwartz RH: Allogeneic non-T spleen
cells restore the responsiveness of normal T cell clones stimulated with
antigen and chemically modified antigen-presenting cells. J Immunol
140:3324, 1988
2. Jenkins MK, Schwartz RH: Antigen presentation by chemically
modified splenocytes induces antigen-specific T cell unresponsiveness
in vitro and in vivo. J Exp Med 165:302, 1987
3. Mueller DL, Jenkins MK, Schwartz RH: Clonal expansion versus
functional clonal inactivation: A costimulatory signalling pathway
determines the outcome of T cell antigen receptor occupancy. Annu Rev
Immunol 7:445, 1989
4. Boussiotis VA, Freeman GJ, Gribben JG, Nadler LM: The role of
B7-1/B7-2:CD28/CLTA-4 pathways in the prevention of anergy, induction of productive immunity and down-regulation of the immune
response. Immunol Rev 153:5, 1996
5. Borriello F, Oliveros J, Freeman GJ, Nadler LM, Sharpe AH:
Differential expression of alternate mB7-2 transcripts. J Immunol
155:5490, 1995
6. Linsley PS, Brady W, Grosmaire L, Aruffo A, Damle NK,
Ledbetter JA: Binding of the B cell activation antigen B7 to CD28
costimulates T cell proliferation and interleukin 2 mRNA accumulation.
J Exp Med 173:721, 1991
7. Gimmi CD, Freeman GJ, Gribben JG, Sugita K, Freedman AS,
Morimoto C, Nadler LM: B-cell surface antigen B7 provides a
costimulatory signal that induces T cells to proliferate and secrete
interleukin 2. Proc Natl Acad Sci USA 88:6575, 1991
8. Freeman GJ, Gray GS, Gimmi CD, Lombard DB, Zhou LJ, White
M, Fingeroth JD, Gribben JG, Nadler LM: Structure, expression, and T
cell costimulatory activity of the murine homologue of the human B
lymphocyte activation antigen B7. J Exp Med 174:625, 1991
9. Freeman GJ, Gribben JG, Boussiotis VA, Ng JW, Restivo VA, Jr,
Lombard LA, Gray GS, Nadler LM: Cloning of B7-2: A CTLA-4
counter-receptor that costimulates human T cell proliferation [see
comments]. Science 262:909, 1993
10. Chen C, Faherty DA, Gault A, Connaughton SE, Powers GD,
Godfrey DI, Nabavi N: Monoclonal antibody 2D10 recognizes a novel
T cell costimulatory molecule on activated murine B lymphocytes. J
Immunol 152:2105, 1994
11. Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T
cell costimulation. Annu Rev Immunol 14:233, 1996
12. Lenschow DJ, Su GH, Zuckerman LA, Nabavi N, Jellis CL,
Gray GS, Miller J, Bluestone JA: Expression and functional significance of an additional ligand for CTLA-4. Proc Natl Acad Sci USA
90:11054, 1993
13. Hathcock KS, Laszlo G, Pucillo C, Linsley P, Hodes RJ:
Comparative analysis of B7-1 and B7-2 costimulatory ligands: Expression and function. J Exp Med 180:631, 1994
14. Lenschow DJ, Sperling AI, Cooke MP, Freeman G, Rhee L,
Decker DC, Gray G, Nadler LM, Goodnow CC, Bluestone JA:
Differential up-regulation of the B7-1 and B7-2 costimulatory mol-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MOLECULAR REGULATION OF B7-2
ecules after Ig receptor engagement by antigen. J Immunol 153:1990,
1994
15. Daikh D, Wofsy D, Imboden JB: The CD28-B7 costimulatory
pathway and its role in autoimmune disease. J Leukoc Biol 62:156,
1997
16. Boehm U, Klamp T, Groot M, Howard JC: Cellular responses to
interferon-gamma. Annu Rev Immunol 15:749, 1997
17. Varesio L, Cox GW, Pulkki K, Musso T, Latham PS, Gusella GL,
Espinoza-Delgado I: Activation of macrophages and monocytes by
interferon-gamma and interleukin-2, in Klostergaard J, Lopez-Berestein
G (eds): Mononuclear Phagocytes in Cell Biology. Boca Raton, FL,
CRC, 1993, p 119
18. Espinoza-Delgado I, Longo DL, Gusella GL, Varesio L: Regulation of IL-2 receptor subunit genes in human monocytes. Differential
effects of IL-2 and IFN-gamma. J Immunol 149:2961, 1992
19. Kelley VE, Fiers W, Strom TB: Cloned human interferongamma, but not interferon-beta or -alpha, induces expression of
HLA-DR determinants by fetal monocytes and myeloid leukemic cell
lines. J Immunol 132:240, 1984
20. Littman BH, Dastvan FF, Carlson PL, Sanders KM: Regulation
of monocyte/macrophage C2 production and HLA-DR expression by
IL-4 (BSF-1) and IFN-gamma. J Immunol 142:520, 1989
21. Taetle R, Honeysett JM: Gamma-interferon modulates human
monocyte/macrophage transferrin receptor expression. Blood 71:1590,
1988
22. Taylor LS, Cox GW, Melillo G, Bosco MC, Espinoza-Delgado I:
Bryostatin-1 and IFN-gamma synergize for the expression of the
inducible nitric oxide synthase gene and for nitric oxide production in
murine macrophages. Cancer Res 57:2468, 1997
23. Espinoza-Delgado I, Ortaldo JR, Winkler-Pickett R, Sugamura
K, Varesio L, Longo DL: Expression and role of p75 interleukin 2
receptor on human monocytes. J Exp Med 171:1821, 1990
24. Sen GC, Lengyel P: The interferon system. A bird’s eye view of
its biochemistry. J Biol Chem 267:5017, 1992
25. Philip R, Epstein LB: Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, gammainterferon and interleukin-1. Nature 323:86, 1986
26. Arai KI, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T:
Cytokines: Coordinators of immune and inflammatory responses. Annu
Rev Biochem 59:783, 1990
27. Bosco MC, Espinoza-Delgado I, Schwabe M, Gusella G, Longo
DL, Sugamura K, Varesio L: Regulation by interleukin-2 (IL-2) and
interferon gamma of IL-2 receptor gamma chain gene expression in
human monocytes. Blood 83:2995, 1994
28. Fleischer J, Soeth E, Reiling N, Grage-Griebenow E, Flad HD,
Ernst M: Differential expression and function of CD80 (B7-1) and
CD86 (B7-2) on human peripheral blood monocytes. Immunology
89:592, 1996
29. Ziegler-Heitbrock HW, Thiel E, Futterer A, Herzog V, Wirtz A,
Riethmuller G: Establishment of a human cell line (Mono Mac 6) with
characteristics of mature monocytes. Int J Cancer 41:456, 1988
30. Bosco MC, Rottschafer S, Taylor LS, Ortaldo JR, Longo DL,
Espinoza-Delgado I: The antineoplastic agent bryostatin-1 induces
1789
proinflammatory cytokine production in human monocytes: Synergy
with interleukin-2 and modulation of interleukin-2Rgamma chain
expression. Blood 89:3402, 1997
31. Larsen CP, Ritchie SC, Hendrix R, Linsley PS, Hathcock KS,
Hodes RJ, Lowry RP, Pearson TC: Regulation of immunostimulatory
function and costimulatory molecule (B7-1 and B7-2) expression on
murine dendritic cells. J Immunol 152:5208, 1994
32. Yokozeki H, Katayama I, Ohki O, Arimura M, Takayama K,
Matsunaga T, Satoh T, Umeda T, Azuma M, Okumura K, Nishioka K:
Interferon-gamma differentially regulates CD80 (B7-1) and CD86
(B7-2/B70) expression on human Langerhans cells. Br J Dermatol
136:831, 1997
33. Nikcevich KM, Gordon KB, Tan L, Hurst SD, Kroepfl JF,
Gardinier M, Barrett TA, Miller SD: IFN-gamma-activated primary
murine astrocytes express B7 costimulatory molecules and prime naive
antigen-specific T cells. J Immunol 158:614, 1997
34. DeMaria CT, Brewer G: AUF1 binding affinity to A⫹U-rich
elements correlates with rapid mRNA degradation. J Biol Chem
271:12179, 1996
35. Wagner BJ, Long L, Rao PN, Pettenati MJ, Brewer G: Localization and physical mapping of genes encoding the A⫹U-rich element
RNA-binding protein AUF1 to human chromosomes 4 and X. Genomics 34:219, 1996
36. Hel Z, Skamene E, Radzioch D: Two distinct regions in the 38
untranslated region of tumor necrosis factor alpha mRNA form
complexes with macrophage proteins. Mol Cell Biol 16:5579, 1996
37. Hel Z, Di MS, Radzioch D: Characterization of the RNA binding
proteins forming complexes with a novel putative regulatory region in
the 38-UTR of TNF-alpha mRNA. Nucleic Acids Res 26:2803, 1998
38. Narumi S, Finke JH, Hamilton TA: Interferon gamma and
interleukin 2 synergize to induce selective monokine expression in
murine peritoneal macrophages. J Biol Chem 265:7036, 1990
39. Sariban E, Imamura K, Luebbers R, Kufe D: Transcriptional and
posttranscriptional regulation of tumor necrosis factor gene expression
in human monocytes. J Clin Invest 81:1506, 1988
40. Fenton MJ, Vermeulen MW, Clark BD, Webb AC, Auron PE:
Human pro-IL-1 beta gene expression in monocytic cells is regulated by
two distinct pathways. J Immunol 140:2267, 1988
41. Whittemore LA, Maniatis T: Postinduction repression of the
beta-interferon gene is mediated through two positive regulatory
domains. Proc Natl Acad Sci USA 87:7799, 1990
42. Darnell JEJ, Kerr IM, Stark GR: Jak-STAT pathways and
transcriptional activation in response to IFNs and other extracellular
signaling proteins. Science 264:1415, 1994
43. Heim MH, Kerr IM, Stark GR, Darnell JE, Jr: Contribution of
STAT SH2 groups to specific interferon signaling by the Jak-STAT
pathway. Science 267:1347, 1995
44. Greenlund AC, Morales MO, Viviano BL, Yan H, Krolewski J,
Schreiber RD: Stat recruitment by tyrosine-phosphorylated cytokine
receptors: An ordered reversible affinity-driven process. Immunity
2:677, 1995
45. Kim TK, Maniatis T: Regulation of interferon-gamma-activated
STAT1 by the ubiquitin-proteasome pathway. Science 273:1717, 1996
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1999 94: 1782-1789
Enhanced B7-2 Gene Expression by Interferon-γ in Human Monocytic Cells
Is Controlled Through Transcriptional and Posttranscriptional Mechanisms
R.E. Curiel, C.S. Garcia, S. Rottschafer, M.C. Bosco and I. Espinoza-Delgado
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