Download The Expression of MHC Class II Genes in Macrophages Is Cell

The Expression of MHC Class II Genes in
Macrophages Is Cell Cycle Dependent
This information is current as
of August 3, 2017.
Jordi Xaus, Mònica Comalada, Marta Barrachina, Carmen
Herrero, Eduard Goñalons, Concepció Soler, Jorge Lloberas
and Antonio Celada
J Immunol 2000; 165:6364-6371; ;
doi: 10.4049/jimmunol.165.11.6364
http://www.jimmunol.org/content/165/11/6364
Subscription
Permissions
Email Alerts
This article cites 68 articles, 24 of which you can access for free at:
http://www.jimmunol.org/content/165/11/6364.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
References
The Expression of MHC Class II Genes in Macrophages Is
Cell Cycle Dependent1
Jordi Xaus, Mònica Comalada, Marta Barrachina, Carmen Herrero, Eduard Goñalons,
Concepció Soler, Jorge Lloberas, and Antonio Celada2
T
he expression of several genes correlated with the proliferative state and the cell cycle progression of the cells.
This is a broad group of genes known as the cell cycledependent (CCD)3 genes (1), as they are not expressed, or are
expressed at a very low level, in quiescent nonproliferating cells,
and their expression is markedly increased in growing cells or in
cells stimulated to proliferate. These genes could be grouped in
several categories, such as histones and DNA-synthesizing machinery (histones, thymidine kinase, thymidylate synthase),
growth factors and receptors (IL-2, TGF␤, transferrin receptor),
protooncogenes (c-myc, c-fos, p53, N-ras), and other metabolically
active and structural proteins (calmodulin, hsp70, IFN-␥) (reviewed in Ref. 1).
To ensure proper progression through the eukaryotic cell cycle,
the proteins involved in its regulation must be periodically expressed at appropriate times (2– 4). These proteins, in turn, regulate
other genes whose products mediate mechanical aspects of the
cycle, such as DNA replication. Nevertheless, the regulators of cell
cycle progression, such as cyclins and cyclin-dependent kinases,
apparently affect both basal and activated transcription of genes
not directly involved in cell cycle regulation or cell proliferation
(5, 6). Recently, it has been observed that the cell cycle elements
Departament de Fisiologia (Biologia del Macròfag), Facultat de Biologia and Fundació August Pi i Sunyer, Campus de Bellvitge, Universitat de Barcelona, Barcelona,
Spain
Received for publication June 1, 2000. Accepted for publication August 31, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Comision Interministerial de Ciencia
y Tecnologia (SAF98-102 and PM 98/0200 to A.C.). J.X. is recipient of a fellowship
from the Comissio Interdepartamental de Recerca i Innovacio Tecnologica. M.B. and
M.C. are recipients of fellowships from the Fundacio August Pi i Sunyer.
2
Address correspondence and reprint requests to Dr. Antonio Celada, Departament de
Fisiologia, Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain. E-mail
address: [email protected]
3
Abbreviations used in this paper: CCD, cell cycle dependent; iNOS, inducible NO
synthase; DAPI, 4,6-diamidino-2-phenylindole; CIITA, class II transactivator.
Copyright © 2000 by The American Association of Immunologists
could also affect the translational apparatus and regulate in a CCD
way the translation of several genes (7, 8).
Therefore, there are genes induced in a CCD fashion that are not
involved in cell cycle progression. This is the case of genes that
control some cellular activities. For example, it has been described
that the sensitivity to apoptotic stimuli observed in some cell types
is dependent on the proliferative state of the cells and the progression through the cell cycle (9, 10). Moreover, it has been observed
that the phagocytic capacity of macrophages is up-regulated in the
S phase of the cell cycle (11) and that, during the G2-M phase, the
glucocorticoid receptor is not functionally active in the nucleus
(12, 13). None of these functions are directly related to cell cycle
regulation, but they are regulated in a cell cycle manner.
MHC II molecules are heterodimers that are expressed on the
surface of a limited number of cells and are required to present Ags
to T cells. A lack of class II expression leads to severe immunodeficiency (14), whereas an abnormal expression may cause autoimmune diseases (15). Thus, regulation of the expression of MHC
II molecules is a critical point in the control and maintenance of
the immune response. The expression of MHC class II molecules
in macrophages is induced by IFN-␥ (16).
Most cells of the immune system are quiescent, and their activation induces a massive proliferation. However, macrophages
that are able to proliferate in tissues, thanks to the autocrine production of M-CSF (17, 18), when activated by stimuli such as
IFN-␥ or LPS, block their proliferation and perform their functional activities (19). Due to the duality between proliferation and
activation in macrophages, we were interested in studying how the
proliferative state and the cell cycle position of macrophages at the
time of activation by IFN-␥ could affect their functional activity.
Recently, we have found that macrophages may be affected by
apoptotic stimuli only in some of the cell cycle positions (19).
In these studies, we have used bone marrow-derived macrophages, because they are nontransformed cells that respond to both
proliferative and activating stimuli. We have observed that IFN-␥
induces the expression of MHC class II molecules in macrophages
in a CCD manner. Despite having a higher basal expression of
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Using different drugs, we stopped the cell cycle of bone marrow-derived macrophages at different points. After IFN-␥ stimulation,
macrophages arrested at the G1 phase of the cell cycle did not increase cell surface expression of the MHC class II IA. This
inhibition is specific, because, under the same conditions, IFN-␥ induces the expression of Fc␥ receptors and the inducible NO
synthase mRNA. Treatments that inhibit macrophage proliferation by blocking the cell cycle at the G1 phase, such as adenosine,
forskolin, or LPS, blocked the IFN-␥ induction of IA. Under IFN-␥ treatment, the steady-state levels of IA␣ and IA␤ mRNA did
not increase in cells arrested at the G1 phase and the half-life of the MHC mRNA was not modified. These data suggest that the
cell cycle modulation of IFN-␥-induced MHC II gene expression occurs at the transcriptional level. The expression of the class II
transactivator mRNA induced by IFN-␥ was also blocked when macrophages were arrested at the G1 phase of the cell cycle,
suggesting that the lack of IFN-␥ response occurs at the early steps of MHC class II expression. Finally, macrophages arrested at
the G1 phase showed increased basal levels of cell surface IA due to an increase of the translational efficiency. These data show
that the expression of MHC class II genes is regulated by the cell cycle. The Journal of Immunology, 2000, 165: 6364 – 6371.
The Journal of Immunology
6365
class II molecules, macrophages arrested at the G1 phase of the cell
cycle do not increase class II expression after stimulation with
IFN-␥. This effect is not observed in macrophages arrested at other
phases of the cell cycle and is specific for MHC class II expression.
Moreover, the effect of G1-arrested macrophages on the IFN-␥induced MHC expression is mainly found at the transcriptional
level, whereas the increase of the basal expression is due to an
effect at the translational level.
citric acid, and 2 ␮g/ml DAPI (pH 7.4) was added to each sample. After
incubating the cells at 4°C for 24 h, their fluorescence was measured with
an Epics Elite flow cytometer (Coulter, Hialeah, FL). For this analysis, we
used an UV laser with an excitation beam of 25 mW at 333–364 nm, and
fluorescence was collected with a 525-nm band-pass filter. Cell doublets
were gated out by comparing the pulse area vs the pulse width. For each
histogram, 12,000 cells were counted, and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, San Diego, CA).
Materials and Methods
Total cellular RNA (20 ␮g) was extracted and separated as previously
described (27) in 1% agarose gel with 5 mM MOPS (pH 7.0)/1 M formaldehyde buffer. The RNA was transferred overnight to a GeneScreen nitrocellulose membrane (Life Science Products, Boston, MA) and fixed by
UV irradiation (150 mJ). To check for differences in RNA loading, we
analyzed the expression of 18S ribosomal RNA. All probes were labeled
with [␣-32P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) using the oligolabeling kit method (Pharmacia Biotech, Uppsala, Sweden). After incubating the membranes for 18 h at 65°C in hybridization solution (20%
formamide, 5⫻ Denhart’s, 5⫻ SSC, 10 mM EDTA, 1% SDS, 25 mM
Na2HPO4, 25 mM NaH2PO4, 0.2 mg/ml salmon sperm DNA, and 106
cpm/ml of 32P-labeled probe), they were exposed to Kodak X-AR films
(Kodak, Rochester, NY). The bands of interest were quantified with a
Molecular Imaging System (Bio-Rad, Richmond, CA).
Reagents
Adenosine, 5⬘-N-ethyl-carboxamido-adenosine, mimosine, OH-urea, nocodazole, and LPS were obtained from Sigma (St. Louis, MO). Forskolin
was obtained from Fluka Biochemika (Buchs, Switzerland). [3H]Thymidine was obtained from Amersham International (Buckinghamshire, U.K.).
4,6-diamidino-2-phenylindole (DAPI) was purchased from Calbiochem
(La Jolla, CA). All of the other products were of the best grade available
and were purchased from Sigma. Deionized water further purified with a
Millipore Milli-Q System (Bedford, MA) was used. IFN-␥ was a kind gift
from Genentech (South San Francisco, CA).
Bone marrow-derived macrophages were isolated from 6-wk-old BALB/c
mice (Charles River Breeding Laboratories, Wilmington, MA) as previously described (19, 20). The cells were cultured in plastic dishes (150
mm) in 40 ml DMEM containing 20% FBS and 30% L cell-conditioned
media as a source of M-CSF. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere. After 7 days of culture, a homogeneous
population of adherent macrophages was obtained.
Antibodies
Surface expression of the MHC class II molecules (IAd) was analyzed by
using purified mouse mAb anti-mouse IAd,b (PharMingen, San Diego, CA)
as previously described (21). Fluorescein-conjugated rat anti-mouse IgG
Ab from Cappel (Turnhout, Belgium) was used as a secondary Ab. To
block Fc receptors, we used an anti-CD16/CD32 Ab (PharMingen). The
same Ab was used as primary Ab for the surface analysis of Fc␥ receptor,
and a fluorescein-conjugated anti-rat IgG (Cappel) as a secondary Ab.
Plasmids and constructs
The cDNA probes for IA␣ and IA␤ used for Northern and slot blotting
were a kind gift from P. Cosson (Basel Institute Für Immunobiologie,
Basel, Switzerland) (22). A rat-inducible NO synthase (iNOS) cDNA fragment (23) was used for the detection of IFN-␥-induced iNOS expression.
The class II transactivator (CIITA) probe was made by PCR with the following primers: 5⬘-GACCTGGATCGTCTCGTCCAG and 3⬘-CTCAGC
CTTAGGAGGGACTTG, as indicated by Richard Flavell (Yale University, Howard Hughes Medical Institute, New Haven, CT) (24), that produced a fragment of 355 bp. An 18S ribosomal RNA probe (25) was used
as a control for the amount of loaded RNA.
Determination of cell surface protein expression
Cell surface staining was conducted using specific Abs and cytofluorometric analysis (21, 26). Once arrested at the different cell cycle phases with
the indicated cell cycle inhibitors, macrophages were treated with 300 U/ml
during a pulse of 2 h, and class II surface expression was measured after
24 or 48 h. A total of 106 cells was harvested and washed in cold PBS.
After fixation with 2% paraformaldehyde during 30 min at 4°C, the cells
were resuspended in 50 ␮l PBS containing 5% FBS. In the case of class II
MHC expression, they were then incubated at 4°C for 15 min with 1 ␮g/
106 cells of anti-CD16/CD32 mAb to block Fc receptors. Then the cells
were incubated for 1 h at room temperature with mouse mAb anti-mouse
IAd,b (1 ␮g/106 cells) or directly incubated at 4°C for 60 min with 1 ␮g/106
cells of anti-CD16/CD32 mAb to analyze the Fc␥ receptor expression. The
cells were then washed by centrifugation through a FBS cushion. Finally,
they were incubated with FITC-conjugated secondary Ab for 1 h at 4°C.
Stained cell suspensions were analyzed using an Epics XL flow cytometer
(Coulter). FITC excitation was obtained using a 488-nm argon laser lamp,
and its fluorescence was collected with a 525-nm band-pass filter. The
parameters used to select cell populations for analysis were forward and
side light scatter. A nonrelated Ab was used as a control for nonspecificity.
Polysome gradients
Cell proliferation was measured by using a previously described procedure
(18, 26) with minor modifications. To analyze the effect of LPS and adenosine on macrophage proliferation, the cells were deprived of M-CSF for
18 h and then 105 cells were incubated for 24 h in 24-well plates (Costar,
Cambridge, MA) in 1 ml of complete medium in the presence or absence
of 100 ng/ml LPS, 10⫺5 M forskolin, or 5 ⫻ 10⫺5 M adenosine. Then the
medium was removed and replaced by 0.5 ml of medium containing
[3H]thymidine (1 ␮Ci/ml). After 2 additional h of incubation at 37°C, the
medium was removed and the cells were fixed in ice-cold 70% methanol.
After three washes in ice-cold 10% TCA, the cells were solubilized in 1%
SDS and 0.3% NaOH. Radioactivity was counted by liquid scintillation
using a 1500 Tri-Carb Packard scintillation counter (Packard, Meriden,
CT). Each point was performed in triplicate, and the results were expressed
as the means ⫾ SD.
The cells were collected and washed in ice-cold PBS. The pellet was resuspended in 1 ml of lysis buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl,
1.5 mM Mg Cl2, and 0.5% (v/v) Nonidet P-40) supplemented with 10 ␮l
of RNase inhibitor (RNAguard; Pharmacia Biotech). The cell lysate was
centrifuged for 2 min at 3000 ⫻ g at 4°C. The supernatant was then transferred to a new tube containing heparin to 0.6 ␮g/ml, cycloheximide to
0.15 ␮g/ml, DTT to 20 mM, and PMSF to 1 mM. Finally, the lysate was
centrifuged again for 5 min at 4°C; loaded onto a 10-ml linear 15– 40%
sucrose gradient that had been prepared as described previously (28) in 10
mM Tris-HCl (pH 7.5), 140 mM NaCl, and 1.5 mM MgCl2; and centrifuged for 3 h at 28,000 rpm in a Beckman SW28.1 (Beckman, Fullerton,
CA). Fractions of 550 ␮l were collected into tubes containing SDS to 1%,
EDTA (pH 8) to 10 mM, and proteinase K to 200 ␮g/ml. The fractions
were incubated for 30 min at 37°C, followed by phenol/chloroform extraction and ethanol precipitation. The specific mRNA content of each
fraction was analyzed by slot blotting. The position of ribosomes in the
gradient was assessed by hybridization with an 18S ribosomal RNA probe.
To ensure that the denser fractions contained polysome-bound mRNA, we
prepared sucrose gradients where the 1.5 mM MgCl2 had been substituted
with 10 mM EDTA. In all cases, the mRNA accumulated in the top fractions are polysome-bound (data not shown).
Analysis of DNA content with DAPI
Slot blot
Proliferation assay
6
A total of 10 cells previously subjected to a specific treatment was resuspended and fixed in ice-cold 70% ethanol (19, 26). The cells were then
washed in PBS, resuspended in 0.2 ml of a solution containing 150 mM
NaCl, 80 mM HCl, and 0.1% Triton X-100, and incubated at 4°C for 10
min. Afterward, 1 ml of a solution containing 180 mM Na2HPO4, 90 mM
One-fifth of each fraction of the gradient was applied to the membrane in
5⫻ SSC, 20 mM Tris-HCl (pH 7.5), and 18.5% formaldehyde using a
vacuum manifold (Minifold II; Schleicher & Schüll, Dassel, Germany).
Hybridizations were conducted as described in the section containing
Northern blotting.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
Cell culture
Northern blot analysis
6366
CELL CYCLE-DEPENDENT REGULATION OF MHC CLASS II EXPRESSION
Results
FIGURE 1. G1 arrest inhibits IFN-␥-induced expression of MHC class
II genes in macrophages. A, Mimosine arrest inhibits IA surface expression
in macrophages induced by IFN-␥. A total of 106 cells was treated with 100
ng/ml mimosine, 100 ng/ml OH-urea, or 1 ␮g/ml nocodazole for 16 h (19).
After synchronization, the macrophages were activated with 300 U/ml
IFN-␥ for 2 h. Then the medium was removed and the cells were plated
again in fresh complete media, and MHC class II surface expression was
analyzed at each indicated time. The continuous line histogram shows IA
surface expression in nonstimulated cells, whereas the noncontinuous line
histogram corresponds to IA expression in cells activated with IFN-␥. Fc
receptors were blocked using a specific Ab (anti-CD16/CD32). B, Quantification of the IA surface expression using the Immuno4 software. C,
Northern blot analysis of IA␣ and IA␤ mRNA expression. A total of 20 ␮g
of total RNA from macrophages treated in the same experimental conditions described in A was probed with a randomly primed 32P-labeled IA␣
or IA␤ cDNA fragment. The amount of loaded RNA was corrected by
ribosomal 18S RNA gene expression. The results are representative of
three independent experiments. SD observed between independent experiments was lower than 10% in all cases.
Therefore, the lack of IFN-␥ induction of the MHC class II Ags in
the G1 phase of the cell cycle was specific and not a general inhibition of IFN-␥-induced macrophage activation. These results
also suggested that the effect observed on MHC class II genes was
not due to a reduction in the expression of IFN-␥ receptors on the
surface of macrophages during the G1 phase of the cell cycle or to
a blockage of the signal transduction mechanism. Moreover, the
effect observed in G1-arrested cells could not be related to any
toxic effects of mimosine, because it did not affect the surface
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
In these studies, we have used bone marrow-derived macrophages
because they are primary nontransformed cultures with an unmodified cell cycle machinery that respond to macrophage activators
(IFN-␥, LPS) or proliferate with several growth factors (M-CSF,
GM-CSF, IL-3) (16, 18). Macrophages express low levels of MHC
class II molecules under basal conditions, and they must be activated to induce the expression of MHC class II Ags (16). IFN-␥ is
the major macrophage activator that induces the expression of
MHC class II molecules with a maximal induction 24 – 48 h after
stimulation. Moreover, IFN-␥ also inhibits macrophage proliferation, blocking the cell cycle at the G1-S boundary (19, 26).
To study the effects of the cell cycle on the induction by IFN-␥
of the expression of MHC class II molecules, we first synchronized
macrophages in a reversible way in each of the cell cycle phases.
To this purpose, we used cell cycle inhibitors described elsewhere
(19). The treatment of macrophages growing in the presence of
M-CSF for 16 h with 100 ng/ml mimosine, 100 ng/ml OH-urea, or
1 ␮g/ml nocodazole-arrested macrophages at the G1, S, or G2-M
phases, respectively (19). The concentrations of inhibitors used did
not affect cell viability, as measured by flow cytometry and trypan
blue exclusion. Mimosine is a plant amino acid that inhibits the
initiation of replication and blocks cell cycle in the middle-late G1
phase (29, 30), whereas OH-urea inhibits DNA replication by interfering with DNA polymerases (31) and stops cell cycle at the
beginning of the S phase after crossing the restriction point (32).
Nocodazole inhibits the cell cycle of macrophages at the G2-M
phase by blocking the polimerization of tubuline needed for the
microspindle formation (33, 34).
Macrophages were arrested at each phase of the cell cycle by
treatment with the specific inhibitors for 16 h. Then the cells were
activated with 300 U/ml IFN-␥ for 2 h. After that, the cells were
washed to remove any remaining inhibitors and IFN-␥ and cultured again in fresh complete media for another 24 or 48 h, after
which the expression of MHC class II molecules was determined.
The incubation with saturating amounts of IFN-␥ for 2 h resulted,
24 h later, in a maximal expression of MHC class II molecules on
cell surface that was similar to the level of expression obtained
with a continuous stimulation with IFN-␥ (35). According to this
protocol, the macrophages were first incubated in the presence of
the cell cycle inhibitors alone to arrest all of the cell populations at
the same cell cycle stage. Once the blockage had been established,
the pulse of IFN-␥ was administered. Finally, both the inhibitor
and IFN-␥ were removed and the incubation was resumed in fresh
medium, allowing the cells to fully express the effect of the IFN-␥
pulse on the expression of MHC class II molecules. The surface
expression of MHC Ags was analyzed by flow cytometry using
purified mAbs against IAd molecules. In these conditions, macrophages arrested in the G1 phase did not respond to IFN-␥ and the
expression of MHC molecules on the cell surface was not induced
(Fig. 1, A and B). This correlated with an inhibition of the MHC
mRNA expression induced by IFN-␥ (Fig. 1C). No significant differences in MHC class II-induced expression were observed in
cells arrested at other phases of the cell cycle.
To determine whether the effects of cell cycle on MHC class II
gene expression were a general feature affecting many different
genes, we measured the IFN-␥-induced expression of Fc␥ receptor
under the same conditions. Macrophages arrested at different
points of the cell cycle did not show differences in the IFN-␥induced expression of Fc␥ receptor (Fig. 2).
Besides, we did not detect differences between the IFN-␥-induced expression of iNOS mRNA in macrophages arrested at the
G1 phase with mimosine and in control macrophages (Fig. 3).
The Journal of Immunology
6367
FIGURE 2. Cell cycle does not modify IFN-␥-induced expression of
Fc␥R in macrophages. A, A total of 106 bone marrow-derived macrophage
cells was treated with 100 ng/ml mimosine, 100 ng/ml OH-urea, or 1 ␮g/ml
nocodazole for 16 h. After synchronization, the macrophages were activated with 300 U/ml IFN-␥ for 2 h. Then the medium was removed, and
the cells were plated again in fresh complete media and Fc␥R surface
expression was analyzed after 24 h. The continuous line histogram shows
Fc␥R surface expression in nonstimulated cells, whereas the noncontinuous line histogram corresponds to Fc␥R expression in cells activated with
IFN-␥. B, Quantification of the Fc␥R surface expression after activation
with IFN-␥ using the Immuno4 software. Similar results were obtained in
another independent experiment.
expression of Fc␥ receptors or iNOS mRNA expression. Therefore, we concluded that the cell cycle position could specifically
modulate MHC class II expression on macrophages and its induction by IFN-␥.
Our results so far indicated that cell cycle arrest at the G1 phase
inhibited IFN-␥-induced expression of MHC class II genes. Therefore, those treatments that inhibit macrophage proliferation by
blocking the cell cycle at the G1 phase might be inhibiting MHC
class II expression induced by IFN-␥ in these cells. In fact, it has
been suggested that the agents that increase cAMP levels could be
inhibitors of macrophage activities (21, 36). It has also been reported that LPS inhibits, in some cases, certain aspects of IFN-␥induced macrophage activation, whereas, in other cases, it increases activation (16, 37–39). Both cAMP-increasing agents and
LPS inhibit M-CSF-dependent proliferation of macrophages. The
treatment of macrophages with adenosine, forskolin (Fig. 4A), or
LPS (Fig. 4B) inhibited the [3H]thymidine incorporation induced
by M-CSF completely.
The analysis of DNA content using flow cytometry of DAPIstained cells showed that bone marrow macrophage cultures growing in the presence of M-CSF were not cell cycle-synchronized
and showed a random distribution, with 51% of cells in G0-G1,
30% in S, and 17% in G2-M (Fig. 4C). In response to adenosine,
macrophages appeared to be distributed homogeneously (88% of
total cells) in a peak corresponding to the G1 phase of the cell
cycle. The treatment of macrophages with 100 ng/ml LPS also
induced a G1 arrest of ⬎75% of viable cells, but, in contrast with
adenosine, LPS induced the appearance of a subdiploid peak that
accounts for 35% of the culture and corresponds to cell death by
apoptosis.
As shown by Northern blotting (Fig. 4, D and E), the treatment
of macrophages with cAMP-increasing agents or LPS also inhibited the expression of MHC class II genes induced by IFN-␥.
Therefore, both cAMP-increasing agents and LPS inhibited macrophage proliferation by blocking the cell cycle at the G1 phase,
and all three inhibited the IFN-␥-induced expression of MHC class
II molecules.
It has been described that the cell cycle modulates the expression of several genes not only at the transcriptional level but also
through posttranscriptional mechanisms (6 – 8). To determine
whether the modifications observed in the CCD regulation of
MHC expression are due to transcriptional changes or to the rate of
mRNA degradation, we analyzed the half-life of MHC II transcripts. The rate of degradation was measured after 24 h of IFN-␥
treatment in cells arrested with mimosine or in control cells. Actinomycin D was added to a concentration (5 ␮g/ml) sufficient to
block all further RNA synthesis, as determined by [3H]UTP incorporation (16, 40), and the RNA was isolated at intervals of 2 h
thereafter. IA␤ mRNA was very stable both under the treatment
with mimosine and in control macrophages (Fig. 5), thus suggesting that the lack of IFN-␥ induction of mRNA levels in G1-arrested
cells was due to a lack of transcriptional activation.
Recently, we have shown that IFN-␥ regulates the expression of
MHC class II molecules in bone marrow macrophages not only at
the transcriptional level (16) but also at the translational level (28).
Therefore, we also analyzed whether the blockage effect in IFN␥-induced MHC class II expression observed in G1-arrested cells
could be caused by the translational machinery. As we have reported elsewhere, initiation is the step where translation of IA␣
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 3. G1 arrest does not inhibit IFN-␥-induced expression of
iNOS mRNA in macrophages. The cells were treated or not with 100 ng/ml
mimosine. After 16 h of mimosine treatment, the macrophages were activated with 300 U/ml IFN-␥ for 2 h. Then the medium was removed, and
the cells were plated again in fresh complete media and iNOS mRNA
expression was analyzed at the indicated times (hours) by Northern blotting
as previously described. The amount of loaded RNA was corrected by
ribosomal 18S RNA gene expression. The blots were quantified using a
radioanalytic imaging system (Molecular Analyst System; Bio-Rad). These
results are representative of two independent experiments. SD observed
between independent experiments was lower than 10% in all cases.
6368
CELL CYCLE-DEPENDENT REGULATION OF MHC CLASS II EXPRESSION
FIGURE 5. G1 arrest does not modify the half-life of MHC class II
mRNA. MHC class II expression was induced by treatment with IFN-␥ for
24 h in macrophages growing in the presence of M-CSF (control) or previously synchronized at the G1 phase by treatment with 100 ng/ml mimosine for 16 h. Then the macrophages were treated with actinomycin D
(5 ␮g/ml) to block transcription (40). The samples were obtained at the
indicated times, and the mRNA corresponding to IA␤ was quantified by
Northern blotting. The data are represented as percentage of IA␤ mRNA
after actinomycin D treatment compared with the quantity of IA␤ mRNA
induced before actinomycin D treatment. The same results were found in
two independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 4. Both LPS and adenosine block macrophage proliferation at
the G1 phase and inhibit IFN-␥-induced MHC class II expression. The
agents that increase cAMP (A) and LPS (B) inhibit M-CSF-dependent proliferation of bone marrow-derived macrophages. The macrophages were
obtained after 7 days of culture in the presence of M-CSF. A total of 105
macrophages was incubated in 24-well plates in the presence of the indicated amounts of M-CSF either alone (M-CSF) or with 100 ng/ml LPS,
5 ⫻ 10⫺5 M adenosine or 10⫺5 M forskolin. Proliferation was determined
as indicated in Materials and Methods. Each determination was made in
triplicate, and the values represent the means ⫾ SD. C, Both adenosine and
LPS arrest macrophages at the G1 phase of the cell cycle. A total of 106
macrophages grown in the presence of M-CSF was treated or not with 100
ng/ml LPS or 5 ⫻ 10⫺5 M adenosine for 24 h, and their DNA content was
measured by flow cytometry. D, The agents that increase cAMP levels
inhibit IFN-␥-induced MHC expression. The macrophages were treated
with 30 U/ml IFN-␥ for 24 h in the presence or absence of 5⬘-N-ethylcarboxamido-adenosine (10⫺5 M) or forskolin (10⫺5 M). E, LPS blocks
IFN-␥-induced expression of IA genes in macrophages. The cells were
treated with 30 U/ml IFN-␥ for 24 h in the presence or absence of 100
ng/ml LPS. A total of 20 ␮g of total RNA was used in the Northern blotting
analysis of IA␣ and IA␤ mRNA. The amount of loaded RNA was corrected using the ribosomal 18S RNA gene expression. The membranes
were quantified using a Molecular Imaging System (Bio-Rad). The results
are representative of three independent experiments. SD observed between
independent experiments was lower than 10% in all cases.
and IA␤ is controlled (28). Thus, polysome gradients were obtained and the profiles of IA␣ and IA␤ mRNAs were analyzed
both before and after IFN-␥ treatment in cells arrested or not at G1
with mimosine. Fractionation of the cellular mRNAs in a sucrose
gradient allows the separation of free mRNA from mRNA that is
bound to one or more ribosomes. As seen in Fig. 6, in noninduced
cells, 30 and 24%, respectively, of the IA␣ and IA␤ mRNAs were
free and the rest was evenly distributed along the ribosome-bound
fractions. However, after IFN-␥-stimulation, both IA␣ and IA␤
mRNAs underwent a marked shift toward polysome-bound mRNAs, and only 8 and 7% were free. A similar shift was observed
in G1-arrested cells that had not been stimulated with IFN-␥ (8 and
1%), and this shift was not modified after IFN-␥ activation (10 and
5%). This indicated that the G1 arrest induced an increase in the
average number of ribosomes bound to these specific mRNAs, and
consequently increased protein synthesis efficiency. As a control
we used ␤-actin (28, 40), which did not show any changes in its
distribution profile or in the mean number of ribosomes attached
per molecule of ␤-actin mRNA after treatment with IFN-␥ or with
mimosine. The distribution of 18S mRNA was used to determine
the position of one ribosome (28, 40).
Thus, G1 arrest induced an increase in the efficiency of MHC
class II protein synthesis that may account for the increase of basal
MHC surface expression observed in the G1 phase of the cell cycle. As shown in Fig. 7, when the cells are arrested at the G1 phase
of the cell cycle, there is an increase in the levels of MHC class II
IA molecules on the cell surface. However, translational control
was not involved in the inhibition of IFN-␥-induced MHC class II
expression observed in cells arrested at the G1 phase of the cell
cycle.
The transcriptional regulation of the expression of MHC class II
genes involves at least three cis-acting elements located 5⬘ upstream of the transcription initiation site (reviewed in Refs. 41–
43). These elements have been referred to as W, X, and Y, and NFs
have been shown to bind to each element. The expression of these
transcription factors is ubiquitous and is not regulated by IFN-␥.
However, the correct expression of MHC class II genes requires
the presence of CIITA, a trans-activator that does not bind directly
to the DNA, is tissue specific, and whose expression is regulated
by IFN-␥ in macrophages (44). To determine whether this transcription factor is involved in the CCD expression of MHC class
II molecules, we analyzed the IFN-␥-induced expression of this
gene in macrophages arrested at the different phases of the cell
cycle.
The Journal of Immunology
The steady-state levels of CIITA mRNA in macrophages were
monitored during the treatment of the cells with IFN-␥ (Fig. 8).
The expression of CIITA was detected after 6 h of treatment with
IFN-␥. Macrophages arrested at the G1 phase of the cell cycle after
treatment with mimosine showed a higher basal expression, but no
induction was observed after treatment with IFN-␥. These results
could explain the specific effect of the cell cycle on class II expression and suggested that CCD IFN-␥-induced expression of
MHC class II molecules is regulated mainly at the transcriptional
level.
Discussion
The term CCD genes designates a very broad group of genes that
are expressed in a CCD manner. This means that they are not
expressed, or are expressed at very low level, in quiescent nonproliferating cells, and their expression is markedly increased in
growing cells or in cells stimulated to proliferate. This includes
genes that are directly involved in cell cycle regulation, together
with other genes not involved in cell proliferation and that participate in several signaling cascades (reviewed in Ref. 8). Moreover,
the activity of some elements of the cell cycle machinery may
modulate the expression or the activity of several genes acting on
the transcriptional and/or translational machinery (45–51). Therefore, it is not surprising that the cell cycle position or the proliferative state of a cell could modulate its activity.
FIGURE 7. Macrophages arrested at the G1 phase have an increased
basal expression of MHC class II molecules on the cell surface. A, Mimosine arrest increases basal IA surface expression in bone marrow-derived macrophages. A total of 106 macrophages was treated with 100 ng/ml
mimosine, 100 ng/ml OH-urea, or 1 ␮g/ml nocodazole for 16 h, and MHC
class II surface expression was analyzed by flow cytometry. The continuous line histogram shows basal IA surface expression in nonsynchronized
cells, whereas the noncontinuous line histogram corresponds to IA expression in cells synchronized with each cell cycle inhibitor. The Fc receptors
were blocked using a specific Ab (anti-CD16/CD32). B, Quantification of
IA surface expression using the Immuno4 software. Similar results were
obtained in three independent experiments.
We have observed that IFN-␥ induces the expression of MHC
class II molecules in macrophages in a CCD manner, because macrophages arrested at the G1 phase of cell cycle do not show an
increase in the expression of class II molecules after stimulation
with IFN-␥; however, these cells do have a higher basal expression
of class II molecules. The detectable levels of IA mRNAs as well
as IA proteins on the cell surface are in accordance with previous
models, where residual amounts of class II expression were detected despite a lack of response to IFN-␥. This is the case of the
targeted gene inactivation of CIITA (24) or STAT1 genes (52).
The basal increase found in MHC class II expression in macrophages is in accordance with early studies performed with B
cells (53) or macrophages arrested by M-CSF starvation (54). The
effect on IFN-␥-induced expression observed in G1-arrested cells
is a new observation. This effect is not due to mimosine toxicity for
several reasons: first, mimosine arrest is reversible and does not
modify macrophage viability at the concentrations used; second,
mimosine arrest does not inhibit basal expression but up-regulates
it; and third, IFN-␥-induced surface expression of Fc␥ receptors or
iNOS mRNA are not abolished in the same experimental
conditions.
The effect of G1-arrested macrophages on the IFN-␥-induced
MHC expression takes place mainly at the transcriptional level,
because we have found an inhibition of the CIITA and MHC class
II mRNA IFN-␥-induced expression and we did not detect any
variations in the mRNA half-life. However, the increase in the
basal cell surface expression may be due to posttranscriptional
events, related to the increases translational efficiency. The mechanisms underlying transcriptional repression during the cell cycle
are poorly understood. Different mechanisms of transcriptional repression have been proposed (55), including a direct inhibition of
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 6. Shifts in the polysome gradient distribution profile of IA␣
and IA␤ mRNA in macrophages after IFN-␥ stimulation in control and
G1-arrested cells. The cells were treated with 300 U/ml IFN-␥ for 2 h or left
untreated after G1 synchronization with 100 ng/ml mimosine. After IFN-␥
treatment, the cells were washed and cultured for 48 h more to assess class
II expression. Then they were harvested and the RNA was separated into
fractions in a sucrose gradient. The specific mRNA content of each fraction
was assessed by slot blotting. Hybridization with 18S rRNA was performed
to assess the position of one ribosome (indicated with an arrow). ␤-actin
was used as control and no modifications in the polysome distribution were
observed after treatment with IFN-␥ and/or mimosine. These results are
representative of two independent experiments.
6369
6370
CELL CYCLE-DEPENDENT REGULATION OF MHC CLASS II EXPRESSION
general transcription factors (55–57), a local change in the structure of chromatin near the promoter (58 – 60), and inhibition of
DNA binding by competition or steric hindrance (61, 62).
In some promoters, in vivo footprinting techniques have identified two contiguous regulatory elements, known as the CCD element (CDE) and the cell cycle genes homology region (CHR)
(63– 65). These elements are located near the transcription initiation sites and play a key role in the periodic transcription of the
cdc25C, cdc2, and cyclin A genes (63, 66). CDE and CHR are
bound by a transcriptional repressor during the G0-G1 phase that is
released at the S and G2-M phases (63, 64). Apparently, CDE does
not interfere with basal transcription from the core promoter (64).
This CDE-interacting factor, termed CDI-1 (66), represses the activity of transcription factors with glutamine-rich activation domains, such as NF-Y or Sp-1. There are several putative CDE
boxes, but no CHR boxes, in the promoter of the transcription
factor CIITA (67). Using oligonucleotides covering several CDE
boxes from the CIITA promoter, we found no differences in the gel
retardation assay when nuclear extracts from macrophages treated
with mimosine, nocodazole, or OH-urea were used (data not
shown). The lack of IFN-␥-induction of MHC class II genes when
macrophages are arrested at the G1 phase may be related to factors
that regulate CIITA expression. In fact, it has been reported that
CIITA expression depends on protein synthesis (44), thus suggesting that protein factor(s) need to be synthesized to induce CIITA
expression.
The cell cycle dependency of the IFN-␥-induced MHC class II
expression may have its physiologic consequence and could act as
a mechanism of repression of an excessive macrophage activation
during inflammation and a mechanism of resolution of the immune
response. Both LPS and IFN-␥, the major macrophage activators,
as well as other macrophage activators, inhibit macrophage proliferation at different points in the G1 phase of the cell cycle, and
macrophages arrested at G1 phase do not express MHC class II
Acknowledgments
We thank Jaume Comas and Rosario Gonzalez, responsible for the flow
cytometry facility of the Serveis Cientifico Tècnics of the Universitat de
Barcelona. We thank Dr. P. Cosson from the Basel Institute for Immunology (Basel, Switzerland) for the cDNAs for IA␣ and IA␤ genes and Dr. R.
Flavell from the Yale School of Medicine (New Haven, CT) for the information on the CIITA gene. We also thank Martin Cullell-Young for the
revision of this manuscript.
References
1. Kaczmarek, L. 1986. Biology of disease: protooncogene expression during the
cell cycle. Lab. Invest. 54:365.
2. Sherr, C. J. 1994. G1 phase progression: cyclin on cue. Cell 79:551.
3. Draetta, G. F. 1994. Mamalian G1 cyclins. Curr. Opin. Cell Biol. 6:842.
4. Cobrinik, D., S. F. Dowdy, P. W. Hinds, S. Mittnacht, and R. A. Weinberg. 1992.
The retinoblastoma protein and the regulation of cell cycling. Trends Biochem.
Sci. 17:312.
5. Sanchez, I., and B. Dynlacht. 1996. Transcriptional control for the cell cycle.
Curr. Opin. Cell Biol. 8:318.
6. Shiekhattar, R., F. Mermelstein, R. P. Fisher, R. Drapkin, B. Dynlacht,
H. C. Wessling, D. O. Morgan, and D. Reinberg. 1995. CDK-activating kinase
complex is a component of human transcription factor. Nature 374:283.
7. Hengst, L., and S. I. Reed1996. Translational control of p27Kip1 accumulation
during the cell cycle. Science 271:1861.
8. Pearson, R. B., and G. Thomas. 1995. Regulation of p70s6k/p85s6k and its role
in the cell cycle. Prog. Cell Cycle Res. 1:21.
9. Boehme, S. A., and M. J. Lenardo. 1993. Propriocidal apoptosis of mature T
lymphocytes occurs at S phase of the cell cycle. Eur. J. Immunol. 23:1552.
10. Zhu, L., and C. Anasetti. 1995. Cell cycle control of apoptosis in human leukemic
T cells. J. Immunol. 154:192.
11. Walker, W. S., and R. H. J. Beelen. 1988. Macrophage cell cycling: influence of
a proliferative state on the antibody-mediated activities of rat resident peritoneal
macrophages. Cell. Immunol. 111:492.
12. Hsu, S., M. Qi, and D. B. DeFranco. 1992. Cell cycle regulation of glucocorticoid
receptor function. EMBO J. 11:3457.
13. Hsu, S., and D. B. DeFranco. 1995. Selectivity of cell cycle regulation of glucocorticoid receptor function. J. Biol. Chem. 270:3359.
14. Mach, B., V. Steimle, E. Martinez-Soria, and W. Reith. 1996. Regulation of
MHC class II genes: lessons from a disease. Annu. Rev. Immunol. 14:301.
15. Botazzo, G. F., R. Pujol-Borrell, T. Hanafusa, and M. Feldman. 1983. Role of
aberrant HLA-DR expression and antigen presentation in induction of endocrine
autoimmunity. Lancet 2:1115.
16. Celada, A., M. J. Klemsz, and R. A. Maki. 1989. Interferon-␥ activates multiple
pathways to regulate expression of the genes for major histocompatibility class II
I-A␤, tumor necrosis factor and complement component C3 in mouse macrophages. Eur. J. Immunol. 19:1103.
17. Nathan, C. F. 1987. Secretory products of macrophages. J. Clin. Invest. 79:319.
18. Celada, A., and R. A. Maki. 1992. Transforming growth factor-␤ enhances the
M-CSF and GM-CSF-stimulated proliferation of macrophages. J. Immunol. 148:
1102.
19. Xaus, J., M. Cardó, A. F. Valledor, C. Soler, J. Lloberas, and A. Celada. 1999.
Interferon ␥ induces the expression of p21Waf1 and arrests macrophage cell
cycle preventing induction of apoptosis. Immunity 11:103.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
FIGURE 8. G1 arrest inhibits IFN-␥-induced expression of CIITA
mRNA in macrophages. Bone marrow-derived macrophage cells were
treated or not with 100 ng/ml mimosine. After 16 h of mimosine treatment,
the macrophages were activated with 300 U/ml IFN-␥ for 2 h. Then the
medium was removed and the cells were plated again in fresh complete
media and CIITA mRNA expression was analyzed at the indicated times
by Northern blotting as previously described. The amount of loaded RNA
was corrected by ribosomal 18S RNA gene expression. The blots were
quantified using a radioanalytic imaging system (Molecular Analyst System; Bio-Rad). The results are representative of three independent experiments. SD observed between independent experiments was lower than
10% in all cases.
molecules in response to IFN-␥-activation. This also may help to
explain the differences in the immune response observed against
small vs large overwhelming numbers of Gram-negative bacteria.
Early during infections by small numbers of Gram-negative bacteria, IFN-␥ is produced and should diffuse into adjacent tissues.
On the other hand, LPS and bacterial products should be contained
at the site of infection by neutrophils and resident macrophages.
Macrophages would then encounter first IFN-␥ as they approach
the site of infection, and endotoxin only after they enter yet activated to the site of infection, and the MHC class II expression
could be optimal, permitting Ag presentation and an effective host
defense. However, with overwhelming infections, as occurs during
septic shock (68), the quantities of LPS and other bacterial components are too great and they cannot be contained by local phagocytes. Macrophages migrating toward sites of infection might then
encounter LPS before IFN-␥. In these situations, LPS might block
macrophage cell cycle, and the late IFN-␥ activation will be inefficient and the MHC class II expression insufficient to develop a
correct host defense. This would explain the decreased MHC class
II expression and Ag presentation by macrophages observed in
sepsis on both animal models and clinically.
The Journal of Immunology
45. Dynlacht, B. D. 1997. Regulation of transcription by proteins that control the cell
cycle. Nature 389:149.
46. Cavanaugh, A. H., W. M. Hempel, L. J. Taylor, V. Rogalsky, G. Todorov, and
L. I. Rothblum. 1995. Activity of RNA polymerase I transcription factor UBF
blocked by Rb gene product. Nature 374:177.
47. Whyte, R., D. Trouche, K. Martin, S. Jackson, and T. Kouzarides. 1996. Repression of RNA polymerase III transcription by the retinoblastoma protein. Nature
382:88.
48. Rossignol, M., I. Kolb-Cheynel, and J. M. Egly. 1997. Substrate specificity of the
cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J.
16:1628.
49. Gottesfeld, J. M., V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl. 1994. Mitotic
repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIH component. Science 263:81.
50. Martinez-Balbas, M., A. Dey, S. Rabindran, K. Ozato, and C. Wu. 1995. Displacement of sequence-specific transcription factors from mitotic chromatin. Cell
83:29.
51. Colgan, D. F., K. G. K. Murthy, C. Prives, and J. Manley. 1986. Cell-cyclerelated regulation of poly(A) polymerase by phosphorylation. Nature 384:282.
52. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe,
D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. Targeted
disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in
the JACK-STAT signaling pathway. Cell 84:431.
53. Lanier, L. L., and N. L. Warner. 1981. Cell cycle related heterogeneity of Ia
antigen expression on a murine lymphoma cell line: analysis by flow cytometry.
J. Immunol. 126:626.
54. Willman, C. L., C. C. Stewart, V. Miller, T. Yi, and T. B. Tomasi. 1989. Regulation of MHC class II gene expression in macrophages by hematopoietic colony-stimulating factors (CSF). J. Exp. Med. 170:1559.
55. Johnson, A. D. 1995. The price of repression. Cell 81:655.
56. Sauer, F., J. D. Fondell, Y. Ohkuma, R. G. Roeder, and H. Jäckle. 1995. Control
of transcription by Kruppel through interactions with TFIIB and TFIIE ␤. Nature
375:162.
57. Fondell, J. D., F. Brunel, K. Hisatake, and R. G. Roeder. 1996. Unligated thyroid
hormone receptor ␣ can target TATA-binding protein for transcriptional repression. Mol. Cell. Biol. 16:281.
58. Bunker, C. A., and R. E. Kingston. 1994. Transcriptional repression by Drosophila and mammalian Policomb group proteins in transfected mammalian cells.
Mol. Cell. Biol. 14:1721.
59. Nagy, L., H. Y. Kao, D. Chakrabarti, R. J. Lin, C. A. Hassig, and D. E. Ayer.
1997. Nuclear receptor repression mediated by a complex containing SMRT,
mSin3A ans histone deacetylase. Cell 89:373.
60. Hassig, C. A., T. C. Fleischer, A. N. Billin, S. L. Schreiber, and D. E. Ayer. 1997.
A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Cell 89:341.
61. Levine, M., and J. L. Manley. 1989. Transcriptional repression of eukariotic
promoters. Cell 59:405.
62. Cowell, I. G. 1994. Repression versus activation in the control of gene transcription. Trends Biochem. Sci. 19:38.
63. Zwicker, J., F. C. Lucibello, L. A. Wolfraim, C. Gross, M. Truss, K. Engeland,
and R. Müller. 1995. Cell cycle regulation of the cyclin A, cdc25c and cdc2 genes
is based on a common mechanism of transcriptional repression. EMBO J. 14:
4514.
64. Lucibello, F. C., M. Truss, J. Zwicker, F. Ehlert, M. Beato, and R. Müller. 1995.
Periodic cdc25c transcription is mediated by a novel cell cycle-regulated repressor element (CDE). EMBO J. 14:132.
65. Tommasi, S., and G. P. Pfeifer. 1995. In vivo structure of the human cdc2 promoter: release of a p130 –E2F-4 complex from sequences immediately upstream
of the transcription initiation site coincides with induction of cdc2 expression.
Mol. Cell. Biol. 15:6901.
66. Zwicker, J., and R. Müller. 1997. Cell-cycle regulation of gene expression by
transcriptional repression. Trends Genet. 13:3.
67. Muhlethaler-Mottet, A., Otten, L. A., Steimle, V., and Mach, B. 1997. Expression
of MHC class II molecules in different cellular and functional compartments is
controlled by differential usage of multiple promoters of the transactivator CIITA.
EMBO J. 16:2851.
68. Parrillo, J. E. 1993. Pathogenic mechanisms of septic shock. N. Engl. J. Med.
328:1471.
Downloaded from http://www.jimmunol.org/ by guest on August 3, 2017
20. Celada, A., P. W. Gray, E. Rinderknecht, and R. D. Schreiber. 1984. Evidence for
a ␥-interferon receptor that regulates macrophage tumoricidal activity. J. Exp.
Med. 160:55.
21. Xaus, J., M. Mirabet, J. Lloberas, C. Soler, C. Lluis, R. Franco, and A. Celada.
1999. IFN␥ up-regulates the A2B adenosine receptor expression in macrophages:
a mechanism of macrophage deactivation. J. Immunol. 162:3607.
22. Cosson, P., and J. S. Bonifacino. 1992. Role of transmembrane domain interactions in the assembly of class II MHC molecules. Science 258:659.
23. Cuevas, P., M. Garcia Calvo, F. Carceller, D. Reimers, M. Zazo, B. Cuevas, I.
Muñoz-Willery, V. Martinez-Coso, S. Lamas, and G. Gimenez-Gellego. 1996.
Correction of hypertension by normalization of endothelial levels of fibroblast
growth factor and nitric oxide synthase in spontaneously hypertensive rats. Proc.
Natl. Acad. Sci. USA 93:11996.
24. Chang, C. H., S. Guerder, S. C. Hong, W. van Ewijk, and R. A. Flavell. 1996.
Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression. Immunity 4:167.
25. Torczynski, R. M., A. P. Bollon, and M. Fuke. 1983. The complete nucleotide
sequence of the rat 18S ribosomal RNA gene and comparison with the respective
yeast and frog genes. Nucleic Acids Res. 11:4879.
26. Xaus, J., A. F. Valledor, M. Cardó, L. Marquès, J. Beleta, J. M. Palacios, and
A. Celada. 1999. Adenosine inhibits M-CSF-dependent proliferation of macrophages through the induction of p27Kip1. J. Immunol. 163:4140.
27. Valledor, A. F., J. Xaus, L. Marquès, and A. Celada. 1999. Macrophage colonystimulating factor induces the expression of mitogen-activated protein kinase
phosphatase-1 through a protein kinase C-dependent pathway. J. Immunol. 163:
2452.
28. Goñalons, E., M. Barrachina, J. A. Garcia-Sanz, and A. Celada. 1998. Translational control of MHC class II I-A molecules by IFN-␥. J. Immunol. 161:1837.
29. Lalande, M. 1990. A reversible arrest point in the late G1 phase of the mammalian
cell cycle. Exp. Cell Res. 186:332.
30. Mosca, P. J., P. A. Dijkwel, and J. L. Hamlin. 1992. The plant amino acid
mimosine may inhibit initiation at origins of replication in Chinese hamster cells.
Mol. Cell. Biol. 12:4375.
31. Fox, M. H., R. A. Read, and J. S. Bedford. 1987. Comparison of synchronized
chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin or methotrexate. Cytometry 8:315.
32. Maurer-Schultze, B., M. Siebert, and I. D. Bassukas. 1988. An in vivo study on
the synchronizing effect of hydroxyurea. Exp. Cell Res. 174:230.
33. Tobey, R. A., J. G. Valdez, and H. A. Crissman. 1988. Synchronization of human
diploid fibroblasts at multiple stages of the cell cycle. Exp. Cell Res. 179:400.
34. Kato, Y., and Y. Tsunoda. 1992. Synchronous division of mouse two-cell embryos with nocodazole in vitro. J. Reprod. Fertil. 95:39.
35. Celada, A., and R. A. Maki. 1989. The expression of IA correlates with the uptake
of interferon-␥ by macrophages. Eur. J. Immunol. 19:205.
36. Ivashkiv, L. B., and L. H. Glimcher. 1991. Repression of class II major histocompatibility complex genes by cAMP is mediated by conserved promoter elements. J. Exp. Med. 174:1583.
37. Gao, J., D. C. Morrison, T. J. Parmely, S. W. Russell, and W. J. Murphy. 1997.
An interferon-␥-activated site (GAS) is necessary for full expression of the mouse
iNOS gene in response to interferon-␥ and lipopolysaccharide. J. Biol. Chem.
10:1226.
38. Kovarik, P., D. Stoiber, M. Novy, and T. Decker. 1998. Stat1 combines signals
derived from IFN-␥ and LPS receptors during macrophage activation. EMBO J.
17:3660.
39. Chujor, C. S., L. Klein, and C. Lam. 1996. Selective inhibition of interleukin-1
beta gene expression in activated RAW 264.7 macrophages by interferon-␥. Eur.
J. Immunol. 26:1253.
40. Barrachina, M., E. Gonalons, and A. Celada. 1999. LPS upregulates MHC class
II IA molecules expression in B lymphocytes at transcriptional and at translational level. Tissue Antigens 54:461.
41. Benoist, C., and D. Mathis. 1990. Regulation of major histocompatibility complex class II genes: X, Y and other letters of the alphabet. Annu. Rev. Immunol.
8:681.
42. Glimcher, L. H., and C. J. Kara. 1992. Sequences and factors: a guide to MHC
class II transcription. Annu. Rev. Immunol. 10:13.
43. Ting, J. P., and A. S. Baldwin. 1993. Regulation of MHC expression. Curr. Opin.
Immunol. 5:8.
44. Steimle, V., C. A. Siegrist, A. Mottet, B. Lisowska-Grospierre, and B. Mach.
1994. Regulation of MHC class II expression by interferon-␥ mediated by the
transactivator gene CIITA. Science 265:106.
6371