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,
,§
*
Lineberger Comprehensive Cancer Center,
Department of Medicine,
Department of Microbiology and Immunology, and
§
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-p105/p50, and IB
) in unactivated monocytes
and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55)
and gH (UL75), inhibited the induction of these genes. Next, we
demonstrated that these viral ligands directly up-regulated monocyte
gene expression upon their binding to their appropriate cellular
receptors. We then investigated if HCMV binding also resulted in the
translation and secretion of cytokines. Our results showed that HCMV
binding to monocytes resulted in the production and release of IL-1ß
protein. Because these induced gene products have NF-B sites in
their promoter regions, we next examined whether there was an
up-regulation of nuclear NF-B levels. These experiments showed that,
in fact, NF-B was translocated to the nucleus following viral
binding or purified viral ligand binding. Changes in IB levels
correlated with the changes in NF-B translocation. Lastly, we
demonstrated that p38 kinase activity played a central role in IL-1ß
production and that it was rapidly up-regulated following infection.
These results support our hypothesis that HCMV initiates a signal
transduction pathway that leads to monocyte activation and pinpoints a
potential mechanism whereby HCMV infection of monocytes can result in
profound pathogenesis, especially in chronic inflammatory-type
conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we hypothesized that the hallmark of HCMV infection—the
induction and dysregulation of the tightly regulated cellular
transcription factor system (4, 26, 27, 28, 29, 30, 31, 32)—was responsible for the wide
range of clinical symptoms and viral pathogenesis observed in
HCMV-infected individuals (4, 14, 32). We and others have demonstrated
that infection results in the rapid mobilization of NF-B to the
nucleus and increases the expression of the two NF-B subunits,
p105/p50 and p65, in human fibroblasts (27, 29, 32, 33) and that these
changes were mediated initially by the binding of the major HCMV
envelope glycoproteins, gB (UL55) and gH (UL75), to their cognate
cellular receptors (32). This initial cellular activation occurred in a
protein synthesis-independent manner (27, 29, 32). In addition, we
showed that beginning at early times after infection the promoters for
p105/p50 and p65 were transactivated in a protein synthesis-dependent
manner and that the major viral IE gene products along with the induced
cellular factors (such as Sp1) played a role in this transactivation
(26, 27).
The importance of the induction of these host cell factors for the
virus stems from their critical role during infection in the regulation
of the major IE promoter (MIEP), the regulation of the IE genes, as
well as the regulation of vital cellular genes, and, consequently, the
entire gene cascade (26, 27, 30, 34, 35). Specifically, NF-B binds
to and is essential for the transactivation of the MIEP (30, 35). The
essential role for NF-B in the viral life cycle is further
underscored by the fact that, to date, no other documented
stimulus induces the normally constitutively expressed p65 gene
product (27, 33).
In addition to the up-regulation of the viral life cycle, these documented viral-mediated changes in cellular factors would have profound effects on the infected host, because the induced transcription factors would up-regulate a wide array of important cellular genes (not only the ones needed by the virus). This might be particularly important during infection of nonpermissive cells, such as monocytes where all cells can be infected (9), but where viral gene expression is usually limited to only the IE genes (10, 12). Thus, the importance of understanding the up-regulation of these transcription factors and their ultimate downstream targets not only would reveal the overall regulation of the viral life cycle, but also their potential role in viral pathogenicity. As mentioned, we hypothesized that the induction of host cell factors may be central to disease progression in HCMV-infected individuals, and that this would be especially true of immune cells. Therefore, in our next line of study we wanted to determine the effect of viral infection on monocytes because of the physiological role infection of these cells plays in HCMV-associated diseases (4, 6, 14). In particular, we wanted to address the regulation of monocyte activation following viral binding. Thus, we chose to examine the possibility that viral binding to these cells through the major HCMV glycoproteins, gB and gH, could up-regulate the activity of these cells, a scenario that could be envisioned to occur in vivo during cases of active HCMV viremia or perhaps in a localized environment in a persistently infected individual. This possibility is supported by the studies that show that the IE events that take place following HCMV infection including Ca2+ flux, phospholipid turnover, induction of second messengers, up-regulation of transcription factors, and induction of the protooncogenes are similar to the events that occur during known receptor/ligand interactions (27, 28, 29, 32, 36, 37, 38).
Here we show that HCMV binding up-regulates the steady-state message
levels of a number of important monocyte mediator genes (IL-1ß, A20,
NF-B-p105/p50, and IB). In addition, we show that viral
binding activates the monocyte because neutralizing Abs block
viral-mediated message induction, and, more importantly, purified viral
glycoprotein ligands (gB and gH) also increase monocyte mediator mRNA.
We also show that the increase in cytokine message expression
correlates with an increase in cytokine secretion. We went on to
investigate the signaling cascade and demonstrated that this
transcriptional up-regulation is probably due to an increase in
cellular transcription factors as viral binding also induces the
nuclear mobilization of NF-B, which has been reported to up-regulate
many of these induced monocyte-mediator genes (39). Lastly, we showed
that p38, a mitogen-activated protein kinase (MAPK) family member (40),
is activated by viral binding and is an important player in
viral-mediated IL-1ß message expression. This viral binding-mediated
p38 induction appears to be unique to monocytes, as our concomitant
study in fibroblasts showed no induction of p38 at IE times
postinfection (41). Taken together, these studies support our
hypothesis that viral binding through at least the gB and gH
glycoproteins is a bona fide mechanism used by the virus to activate
monocytes. Furthermore, a distinct HCMV-induced pathobiology could be
mediated at least in part by this interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh peripheral blood monocytes from random donors were purified by double density gradient centrifugation as described previously (42). The donors used were HCMV seronegative as determined by ELISA; although under our conditions tested, the HCMV status of the donor had no effect on the parameters we measured. Briefly, whole blood was collected by venipuncture, diluted in RPMI 1640, and the mononuclear cells separated from the RBCs and neutrophils by centrifugation through a Ficoll Histopaque 1077 (Sigma, St. Louis, MO) gradient. The monocytes were then repeatedly washed with ice-cold isotonic saline to remove platelets. Monocytes were further purified from the contaminating lymphocytes by centrifugation through a Percoll (Pharmacia, Uppsala, Sweden) gradient. The cells were then counted and treated as described in the experimental results section.
HCMV infection
Methods for the culturing of our HCMV Towne strain (passage 43–44) in human fibroblasts have been described (27). Monocytes were infected with only gradient-purified virus at a multiplicity of infection (M.O.I.) of 2–3 and incubated at 37°C in 1% human serum in endotoxin-free RPMI 1640 in 6% CO2 incubators for the lengths of time described. HCMV was gradient purified and used to infect cells as previously described (32). UV-inactivated virus was prepared as previously described (31, 32) and was used in the same manner as "live" virus. The UV-inactivated virus did not replicate or produce any detectable levels of IE gene products (data not shown). For the studies in which inhibitors of the various kinases were used, monocytes were pretreated for 1 h before the addition of virus. The inhibitors used were SB203580 (Calbiochem, San Diego, CA), which has been previously shown to inhibit p38 activation (40) and to block HCMV (41) and HIV (43) replication, and PD98059 (Calbiochem), which has been previously shown to inhibit MAP kinase kinase (MEK) activity (44).
Abs and cellular activation
We used the same protocol for the use of the various Abs or
purified ligands in this manuscript as we previously described (32).
For the various experiments involving neutralizing Abs, virus
(gradient-purified "live" or UV-irradiated virus) was preincubated
with a protein G affinity-purified murine monoclonal anti-gB (4
µg of 15D8 (45) of the IgG2A isotype) and anti-gH Ab
(4 µg of 1G6 (46) of the IgG2A isotype) or a protein G
affinity-purified murine monoclonal control Ab (4 µg of an
anti-human prostate-specific Ag (PSA) Ab (anti-PSA or PSA
(of the IgG2A isotype)) for 1 h at 4°C. The 15D8 and
1G6 Abs were a generous gift from Dr. L. Rasmussen (Division of
Infectious Diseases, Stanford School of Medicine, Stanford, CA). For
neutralization with the 1G6 monoclonal Ab, 2% guinea pig complement
(Accurate Scientific, Westbury, NY) was also added. Complement alone
had no effect on monocytes (data not shown). The neutralizing Abs used
in the experiments prevented viral-mediated cytopathic effects and
viral gene expression in fibroblasts as confirmed by side-by-side
experiments in which these variables were assessed (data not shown). In
addition, purified viral gB (1, 20, and 100 ng/ml (32)) or purified
viral gB blocked with Abs (4 µg of 15D8 or the control Ab) were used
in some studies to stimulate cells. In other studies, 15 µg of the
4-3-5 affinity-purified murine anti-idiotypic (Id) IgM Ab (47, 48), which mimics the HCMV viral gH glycoprotein and is thus specific
for the 92.5-kDa cellular gH receptor (49), or 15 µg of an IgM
control Ab (affinity-purified mouse IgM (Southern Biotechnology
Associates, Birmingham, AL)) plus 3 µg of an appropriate
cross-linking Ab (goat anti-mouse IgM Ab (Southern Biotechnology
Associates)) was used to stimulate cells as previously described (32, 38, 50). The 4-3-5 Ab and the appropriate control anti-serum were a
generous gift from Dr. S. Keay (Research Services, The Department of
Veteran Affairs Medical Center, University of Maryland School of
Medicine, Baltimore, MD). In all cases of cellular stimulation
(purified virus, purified gB, Id Ab, or the various control
products), the cells were treated the same (washed identically,
incubated in an equal volume of product and media, incubated for the
same length of time, and harvested in the same manner) to allow for
comparisons between groups. In all cases, samples labeled "Mock"
were treated in a similar manner to the test samples (except no virus,
purified viral ligands, etc. were added), and they were incubated for
the length of the stated experiment. In addition, in some experiments
monocytes were treated with 10 ng/ml of PMA or 1 µg/ml LPS as a
positive control (42, 51). All experiments were repeated.
RNA isolation and Northern blot analysis
Total cellular RNA was collected in guanidinium isothiocyanate
and isolated by cesium chloride centrifugation (42). Then, 5 µg of
total cellular RNA was electrophoresed on a 1% denaturing formaldehyde
agarose gel and transferred overnight to nitrocellulose (Immobilon-NC;
Millipore, Bedford, MA). Expression of specific messages were detected
by Northern blot hybridization. RNA levels were equivalent based on 18S
and 28S rRNA levels. All data presented in this manuscript are from
multiple probes of the same blot; however, all experiments were
repeated two to five times. Nitrocellulose blots were probed with
nick-translated (Boehringer Mannheim, Indianapolis, IN)
32P-labeled cDNA sequences specific for p105/p50 (52), p65
(53), IB (54), IL-1ß (42), and A20 (42). The blots were
hybridized overnight in a 50% formamide solution at 42°C. The blots
were then washed to a stringency of 0.2x SSC at 56°C and developed
for 1–6 days with intensifier screens at -70°C.
ELISA
Monocyte supernatant was harvested following the treatment of cells as stated in the results section. The supernatant was centrifuged three times to remove any cellular debris and then frozen at -70°C until needed. The ELISA was then performed using the manufacturers protocol (Endogen, Cambridge, MA). The samples were run in duplicate along with the appropriate IL-1ß standards. Experiments were repeated and the data shown is from representative experiments.
Cytoplasmic and nuclear extract isolation
Isolation of nuclear extracts have been described elsewhere (27, 33). Briefly, collected cell pellets were incubated for 4 min on ice with a cytoplasmic isolation buffer (10 mM HEPES, pH 7.6, 60 mM KCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF (Sigma), 2 mM phenanthroline (Sigma), 250 µM dichloroisocoumarin (Sigma), 100 µM E-64 (Sigma), and 10 µM pepstatin A (Sigma)). After collection by centrifugation, the nuclear pellets (the cytoplasmic extracts were clarified by centrifugation and stored at -70°C and then used in Western blot analyses) were washed in a cytoplasmic isolation buffer without Nonidet P-40, spun, and then incubated for 10 min on ice with a nuclear isolation buffer (20 mM Tris-HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF (Sigma), 25% glycerol, 2 mM phenanthroline (Sigma), 250 µM dichloroisocoumarin (Sigma), 100 µM E-64 (Sigma), and 10 µM pepstatin A (Sigma)). Supernatants containing the nuclear extracts were collected and stored at -70°C.
Electrophoretic mobility shift assay (EMSA)
Collected nuclear extracts were incubated for 15 min in an EMSA
buffer containing: a binding buffer (10 mM Tris-HCl, pH 7.9, 50 mM
NaCl, 0.5 mM EDTA, 10% glycerol, and 1 mM DTT); 7.5 mM
MgCl2; 0.1 µg polydeoxyinosinic-deoxycytidylic acid
(dIdC); and a 32P-labeled double-stranded
oligonucleotide probe containing a wild-type MHC class I B binding
site (5'-CCTTTTTTTTTGGGGATTCCCCA-3') or a wild-type activation
transcription factor (ATF)/cAMPresponse-element-binding
protein (CREB) binding site
(5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') for experiments
examining NF-B activity and ATF/CREB activity, respectively. The
annealed double stranded oligo probes with "T" overhangs and
"C" ends were labeled by filling in the recessed 3' ends of the
oligo with [-32P]dATP (ICN, Irvine, California) using
Klenow (Boehringer Mannheim), followed by a chase with cold dATP and
dGTP, and then finally G-25 Sephadex (Boehringer Mannheim) column
purified. The samples were electrophoresed on a 5% polyacrylamide gel,
dried, and developed with intensifier screens at -70°C. Abs were
used to supershift the specific complexes of interest by pretreating
the extracts for 15 min to 1 h at 4°C with 1 µg of Ab before
their addition to the binding buffer, MgCl2, dIdC, and
labeled probes. Specific Ab to p50 and p65, as well as preimmune serum,
and their appropriate blocking peptides (a generous gift from Dr.
A. S. Baldwin, Jr., Lineberger Cancer Center, University of
North Carolina, Chapel Hill, NC) were used in the supershift
experiments. Preimmune serum and blocking peptides were used to show
the specificity of the super shifts (data not shown).
Western blot analysis
For the IB and ß Western blot analyses, cytoplasmic
proteins were used and were harvested as stated above. For the p38
Western blot analyses, whole cell extract was used. The various
harvested proteins were collected and added to an SDS-PAGE sample
buffer, boiled, and then stored at -70°C. Equal protein amounts were
added to each lane. The samples were run on a 12% SDS-PAGE gel and
transferred to nitrocellulose (Immobilon-P; Millipore) overnight. The
blots were blocked for 1 h in a 5% skim milk, 0.1% Tween 20, PBS
solution for 1 h, and then incubated with the primary Ab (1:2000
dilution of the anti-IB and anti-IBß Abs; Santa Cruz
Biotechnology, Santa Cruz, CA; or 1:1000 dilution of the
phospho-specific anti-p38 MAPK Ab; New England Biolabs, Beverly,
MA) for 1 h in the same blocking solution. The blots were washed,
incubated for 1 h with a 1:2500 dilution of goat
anti-rabbit-horseradish peroxidase-conjugated secondary Ab in the
same blocking solution, washed again, and finally incubated with the
developing agents and developed as per the enhanced chemiluminescence
protocol (Amersham Life Sciences, Arlington Heights, IL or New England
Biolabs).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the role of HCMV binding and infection in the
activation of monocytes, we initially determined whether there was an
up-regulation of monocyte mediators following infection of monocytes
with HCMV. As seen in Fig. 1
, isolated
nonadherent (NAD) monocytes showed a dramatic increase in the
steady-state mRNA expression of various monocyte effectors (IL-1ß,
A20, p105/p50, and IB) following a 4-h viral infection (with
gradient-purified virus) or incubation with known activating agents (10
ng/ml of PMA or 1 µg/ml of LPS) as compared with the unstimulated
control ("NAD"). Expression of p65, an NF-B subunit, appeared to
change only slightly during the treatments shown in Fig. 1. This was
the only gene product tested that showed donor variability; some donors
showed no effect and some donors showed a small change in expression.
This in itself is not surprising, as p65 message expression is not
induced by any known stimulus except HCMV infection and usually only
occurs at later times postinfection (27). In addition, we also examined
if monocyte adhesion (a known activating stimuli (37, 51), a process
that occurs during extravasation of monocytes from the blood to an
inflammatory site (37, 51, 55, 56), and a process that has hypothesized
to be affected by HCMV infection (57, 58, 59)) was affected by HCMV
infection. As seen in Fig. 1, monocyte adhesion ("ADH") caused an
increase in message expression and HCMV infection further enhanced the
signal ("ADH+HCMV"). Furthermore, it was shown that viral infection
can up-regulate at least IL-1ß expression as early as 30 min to
1 h postinfection (data not shown). Interestingly, this time frame
is similar to the induction of IL-1ß and other monocyte regulatory
products following adhesion to extracellular matrix components or
treatment of monocytes with Abs to various adhesion receptors
(42, 60, 61).
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Next, we wanted to narrow down the nature of the viral-mediated
induction of monocyte gene products. Previously, we showed that a
receptor/ligand-mediated process was responsible for transcription
factor induction in human fibroblasts following viral binding (32).
Therefore, we decided to test if this type of process—a
receptor/ligand-mediated event—was responsible for the activation of
monocytes as measured by an increase in mRNA expression. To demonstrate
that viral binding mediated cellular activation, we examined the role
of the two major HCMV glycoproteins, gB and gH, by preincubating the
gradient-purified virus with neutralizing monoclonal Abs (4 µg of
anti-gB [15D8 (45)] and anti-gH [1G6 (46)]) or a species-
and isotype-matched control Ab (4 µg of anti-PSA (32)) for 60 min
before the addition of the virus to the cells. As shown in Fig. 2, an increase in message expression
following the 4-h incubation of cells with virus was observed (compare
the untreated lanes "NAD" to the infected lanes "NAD+HCMV"). In
contrast, when the virus was preincubated with neutralizing Abs, the
increase in IL-1ß, A20, p105/p50, p65, and IB expression was
blocked (the "NAD+HCMV/gBgH" lane), while the control Ab had no
effect on signaling (the "NAD+HCMV/PSA" lane). In comparison
experiments, the neutralizing function of these Abs was shown to
prevent cytopathic effects and viral gene expression in human
fibroblasts (results not shown). It is important to note that
neutralizing Abs to gB or gH alone can block viral-mediated signaling
in human fibroblasts, while nonneutralizing Abs have no effect (32). We
also wanted to show that UV-irradiated virus, which can bind to cells
but cannot replicate, could activate monocytes. As shown in Fig. 2, irradiated virus also increased the expression of the examined gene
products (the "NAD+UV-HCMV" lane). This signaling was also blocked
by the anti-gB and -gH neutralizing Abs in the same manner as was
seen with "live" virus (data not shown). Thus, these results
support the hypothesis that viral binding itself is sufficient to
activate monocyte gene expression.
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The results from Figs. 1
and 2, as well as our previous results
(32), present the possibility that the initial HCMV-mediated cellular
activation involves a viral-ligand/receptor interaction. Therefore, we
next addressed whether our purified HCMV gB (32) also up-regulated the
expression of the different monocyte gene products. Purified HCMV gB
was incubated under similar conditions to that used for viral
infections. A 4-h incubation of various concentrations of purified HCMV
gB (1, 20, and 100 ng/ml) with the isolated monocytes (lanes marked
"NAD+gB") induced IL-1ß, A20, p105/p50, and IB expression
activity above that seen in the control untreated "NAD" monocytes
(Fig. 3). The 20-ng/ml concentration of
purified gB showed the largest increase in message expression. However,
this amount of gB did not show the largest increase in IL-1ß protein
secretion (see Fig. 5B). As with the use of all human
donors, there is variability, and we attribute these sort of subtle
differences to donor variability. In addition, we used a control lysate
(labeled as "Control"), which was isolated from extracts from the
parental 293T cells (no transfected gB) by the same procedure used to
purify the gB. This control lysate (100 ng/ml) showed no ability to
activate cellular transcription factors, thus demonstrating that the
protocol used to purify gB did not in itself have a mitogenic effect.
Furthermore, the effects mediated by the purified gB (100 ng/ml) could
be blocked by pretreatment with anti-gB Ab (15D8; compare lanes
treated with Ab, "gB", to those without), but not by
pretreatment with an isotype-matched control Ab ("PSA"). The
activation of monocytes following incubation with purified gB and its
inhibition by an anti-gB Ab demonstrates that the specific
gB/receptor interaction directly initiates the signaling process.
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Id Ab, which mimics the HCMV gH
glycoprotein activated monocytes
To show that the viral gH product also played a role in monocyte
activation, we used a well-characterized affinity-purified monoclonal
Id Ab (38, 47, 48, 49, 50), which mimics the HCMV gH glycoprotein and binds
to the same 92.5-kDa cell membrane protein as affinity-purified gH
(49), to stimulate our purified monocytes. We incubated the cells under
the same conditions as with purified gB or "live" or UV-irradiated
virus. The Id Ab (4-3-5) or the appropriate control (a nonspecific
IgM) were preincubated for 60 min with a cross-linking (X-L) secondary
Ab before their 4-h incubation with monocytes. As shown in Fig. 4, gH receptor engagement (lanes marked
with "NAD/Id+X-L") could up-regulate IL-1ß, A20, p105/p50, and
IB expression, while the control Abs had no effect
("NAD/IgM+X-L" or "X-L" alone), thus demonstrating the
specificity of the gH-mediated activation via the Id Ab. The
positive control, monocytes treated with LPS, also demonstrated an
induction of monocyte gene products.
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Next, we wanted to examine if the increase in steady-state mRNA of
the monocyte mediators (specifically IL-1ß) correlated with an
increase in the production and secretion of proteins. To accomplish
this goal, supernatants were harvested from monocytes following their
treatment for 4 h with the various stimulatory products
("live" or UV-inactivated virus or the purified viral ligands) and
then examined by ELISA for IL-1ß levels. As shown in Fig. 5A, HCMV infection as well as
treatment with UV-irradiated virus significantly increased IL-1ß
protein levels. In addition, neutralizing Abs blocked this increase
("gBgH"), while the isotype- and species-matched control Ab
("PSA") had no effect. Treatment of monocytes with our purified
gB (32) increased IL-1ß levels in a dose-dependent fashion (1, 20,
and 100 ng/ml; Fig. 5B). In contrast, treatment of cells
with the control lysate (100 ng/ml) or purified gB (100 ng/ml)
pretreated with an anti-gB Ab did not cause an increase in secreted
IL-1ß levels. The control Ab ("PSA") had no effect. Treatment
of monocytes with an Id Ab ("Id+X-L"), which mimics gH, also
could increase the secretion of IL-1ß in treated cells (Fig. 5C), while the control Ab ("IgM+X-L") had no effect.
Viral infection of adherent monocytes also resulted in an increase in
IL-1ß levels (Fig. 5D). Monocytes were infected at the
time of adherence to tissue culture plates. It can be seen that there
is a large increase in IL-1ß levels in 24-h and 48-h infected cells,
with no activity seen in 4-h infected cells. It should be noted that in
at least one donor there was an increase in IL-1ß protein secreted at
4 h after virus addition (data not shown). This difference in the
regulation of protein secretion in adherent vs nonadherent cells is not
surprising as it has been previously documented to be differences in
the signaling in these cells under these different conditions
(42, 51).
HCMV binding up-regulates NF-B activity in monocytes
Because many of the gene products (IL-1ß, A20, p105/p50, and
IB) that we examined contain NF-B binding sites in their
promoter regions and we are very interested in viral-mediated signal
transduction, we next addressed if NF-B activity was also
up-regulated in monocytes following viral binding. We examined by EMSA
the induction of monocyte NF-B activity following HCMV infection
(gradient-purified "live" or UV-inactivated virus) or incubation
with purified viral ligands for 1 h (Fig. 6A). The results of this study
showed that there was an increase in NF-B DNA binding activity
following HCMV infection (compare lanes 1 and 2)
and following the incubation of monocytes with our purified gB (compare
lanes 1 and 4). In contrast, the control lysate
had no effect on NF-B activity (lane 5).
UV-inactivated virus (lane 3) also up-regulated
NF-B activity. In addition, the viral-mediated induction of NF-B
could be blocked by neutralizing Abs to gB and gH (the control Ab had
no effect (compare lanes 6, 8, and
9)). Lastly, we showed that an Id Ab (which mimics gH)
also stimulated NF-B activity in monocytes (compare lanes
6, 10, and 11). These results support our
work using fibroblasts in which we reported an increase in nuclear
NF-B activity following the engagement of the HCMV glycoproteins gB
and gH with their cellular receptors (32). Furthermore, we also
examined in the same nuclear extracts the induction of ATF/CREB DNA
binding activity, which only was reported to be increased at late times
of infection (62), to demonstrate the specificity of the transcription
factor induction (Fig. 6B). The results of these experiments
showed that ATF/CREB binding activity was not induced during IE times
of infection in monocytes. As shown in Fig. 6C,
supershifting Abs showed that the shifted bands from Fig. 6A
were "classic" NF-B (p50/p65) heterodimers and p50/p50
homodimers (supershifted bands marked with an asterisk). We also used
appropriate blocking peptides and preimmune serum to show the
specificity of the supershifting Abs (data not shown). This rapid
induction of NF-B by HCMV fits our model of the
viral-ligand/cellular-receptor-mediated process being responsible for
an early step of cellular activation, as well as confirms the results
of the induction of NF-B-inducible genes.
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B regulation correlates with NF-B induction in activated
monocytes
To further correlate the increase in NF-B that was seen above,
we also examined the cytosolic lysates for IB and IBß
expression following various treatments of monocytes (Fig. 7). As seen in Fig. 7A showing
a Western blot analysis of IB protein levels, there was a
significant decrease in IB levels following cellular activation
(compare lanes 2, 3, 4, 7,
9, and 10), while there was no change in IB
levels in the control-treated cells (lanes 1,
5, 6, 8, and 11). Fig. 7B shows a graph of the levels of IB as measured by
densitometry. We also examined changes in IBß levels under the
various activating conditions. As seen in Figs. 7, C and
D (Western blot and densitometry analyses), there was no
detectable change in IBß levels. These results are thus consistent
with the virus acting as an acute activating signal because previously
described agents that act as acute activating signals have been shown
to only down-regulate IB, not IBß (39, 63, 64).
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To further characterize the viral-mediated signal transduction
cascade in monocytes, we next wanted to focus on the role of the MAPK
family member p38 (40). To initially examine the role of p38, we used
an inhibitor of p38 activity and then looked at monocyte IL-1ß
message expression. Monocytes were pretreated for 1 h with the
inhibitors (20 µM SB203580, a p38 inhibitor (a dose we have shown
inhibits HCMV replication (41) and Shapiro et al. showed blocks HIV
replication (43)), and 20 µM PD98059, a MEK inhibitor (44)) before
their 1-h incubation with gradient-purified virus. Because the
inhibitors were dissolved in DMSO and were used such that the final
concentration of DMSO was 0.1%, an equivalent amount of DMSO was added
to the control groups. In addition, in none of the samples did DMSO or
the various inhibitors affect cell viability (data not shown).
Following incubation with the inhibitors and the virus, monocytes were
harvested and Northern blots performed (Fig. 8). As shown, only the blocking of p38
function had a major effect on IL-1ß expression (>95% inhibition).
The MEK inhibitor only slightly blocked the IL-1ß signal (5–15%
decrease). We also performed titrations and showed that higher doses
had no additional effects and that the drugs effects were titratable
(data not shown). In addition, we also examined these drugs in
fibroblasts at a number of different concentrations for toxicity and
none of the doses used in our experiments were toxic to these cells in
long-term studies. Our finding on the role of p38 in viral-mediated
signaling in monocytes is apparently uniquely monocytic in nature as
parallel experiments in fibroblasts showed no effect (our unpublished
observations).
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Lastly, because the above mentioned data suggested a role for p38
in viral-mediated signaling, we next wanted to examine if HCMV
infection up-regulated p38 activity. Because it is well documented that
phosphorylated p38 correlates with kinase activity (65), we performed
Western blot analyses of whole cell extracts using a phospho-specific
Ab (recognizes phosphorylated Thr180 and
Tyr182). The results as shown in Fig. 9 demonstrate that p38 is phosphorylated
very rapidly (within 15 min). Following induction, phosphorylated p38
levels begin to decrease, and by 4–8 h levels return to mock levels
(data not shown). The time frame of p38 phosphorylation (15 min to
1 h) correlates with our data presented in Fig. 8 in which the
rapid viral-mediated induction of IL-1ß (within 1 h) was blocked
by a p38 inhibitor. Interestingly, in human fibroblasts, p38 is not
induced during this early time frame (41), suggesting a possible
difference in the mechanisms of viral signaling in nonpermissive
(monocytes) and permissive (fibroblasts) cell types.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and Sp1 levels in human
fibroblasts (32). In addition, this work is supported by the many
studies that have shown that viruses, including other members of the
herpesvirus family, bind to known "activational" receptors
(66, 67, 68, 69, 70). Therefore, our current results plus the work from these
previous studies support our hypothesis that viruses have evolved a
common strategy in which activational molecules/receptors are used for
not only viral entry, but also cellular activation and presumably viral
survival.
We showed that there was an increase in the expression of a number of
monocyte mediators (IL-1ß, A20, p105/p50, and IB) as measured
by Northern blot analysis following viral binding. Interestingly, the
results showed that HCMV induced the message for both an NF-B
subunit, p105/p50, and its inhibitor, IB. Because both gene
products are NF-B inducible, these results are not surprising.
However, one should keep in mind that message induction does not mean
the protein is induced or up-regulated, which under our conditions
examined seem to be the case, as IB levels do not return to
untreated levels even by 4 h poststimulation, suggesting that a
potential viral-mediated translational or posttranslational control
exists (perhaps as seen with the hepatitis B virus HBx protein (71),
the human immunodeficiency virus Tat protein (72), or the human T
lymphotropic virus I Tax1 protein (73)).
We showed that HCMV binding induced A20 expression (originally described by Dixit et al. (74), although we originally called this gene product MAD-6 (60)), an anti-apoptotic factor (75). We have previously shown that this gene product was up-regulated in monocytes activated via their adhesion receptors (42) or adhesion itself (60). A20 can block p53-mediated apoptosis (75) and with the extended life cycle of HCMV in human monocytes (7, 8) and the reported induction of p53 (76), the induction of an anti-apoptotic factor might be necessary for viral survival in these cells. Interestingly, the EBV latent membrane protein 1 induces A20 and this protects these cells from p53-mediated apoptosis (77), suggesting that herpesviruses in general might use the anti-apoptotic properties of this gene product for their own survival.
It was also shown that viral binding could enhance gene expression in
simultaneously adherently activated monocytes. Adherence is a known
physiologic signal responsible for monocyte and macrophage activation
(42, 51, 60, 61, 78, 79). This HCMV-induced message expression was
mediated by viral binding as neutralizing Abs to the major HCMV
glycoproteins (gB and gH) blocked this expression, even in adherent
activated monocytes (data not shown). In addition, we also showed that
UV-irradiated virus also up-regulated message expression, demonstrating
that newly synthesized viral gene products were not required for the
signal. This UV-irradiated virus-mediated signaling could also be
blocked by neutralizing Abs. To show that viral binding truly was
inducing mediator mRNA expression, we examined directly the regulation
of monocyte activation by using purified HCMV gB (described previously
(32)) as a stimulus. The results of these studies demonstrated that
there was indeed a viral glycoprotein-mediated signaling event
occurring in monocytes. In addition, a well-characterized Id Ab (32, 38, 47, 48, 49, 50), which mimics gH and has previously been documented to be
involved in fibroblast signaling (38, 50), also up-regulated monocyte
message expression. These combined results again support our hypothesis
of a novel viral binding-mediated signaling event.
Next, we wanted to examine the effects of our activating conditions on monocyte cytokine secretion, because ultimately monocyte function in many cases is measured by its cytokine secretion. As the results showed, HCMV binding, as well as gB- and gH-mediated signaling, all up-regulated the secretion of IL-1ß protein. In addition, HCMV binding also induced IL-1ß secretion in adherently activated monocytes when added at the initial time of adherence. The lack of IL-1ß secretion in only adherent activated monocytes (in the absence of virus or other activating stimuli) is not surprising as it has previously been shown by Haskill et al. (51) that adherent monocytes need two signals to generate the secretion of IL-1ß protein. Adherence only up-regulates message expression, while a second signal such as that provided by a mitogen or cytokine is needed to get the translational up-regulation of IL-1ß protein. These results suggest the possibility that the virus might provide the second signal, much as a cytokine normally would do, and thus might point to at least one of the reasons behind the immune dysregulation seen in HCMV-infected individuals. Interestingly, we previously showed that integrin engagement in nonadherent monocytes only needed one signal to generate IL-1ß secretion (42), a fact that also seems to occur in our system following viral binding.
The gene products described above that were significantly up-regulated
during viral binding or gB or gH-mediated signaling are all
NF-B-inducible gene products. Therefore, to continue our
investigation of the signaling process we next focused on the
regulation of NF-B in monocytes following the various described
treatments. We showed that there was a rapid increase in nuclear
NF-B levels following viral binding or gB- or gH-mediated binding in
monocytes, which confirmed the results of previous studies in
fibroblasts (26, 27, 29, 30, 33). The data demonstrated that the
induction of NF-B EMSA activity was specific, because ATF/CREB EMSA
activity was not induced supporting the results of Kerry et al. who
showed that ATF DNA binding activity was only up-regulated at late
times of infection (62). Analysis of IB protein levels are
consistent with the changes in NF-B DNA binding levels; that is as
NF-B activity increases, IB levels decrease. Interestingly,
even at 1–4 h poststimulation (either "live" or UV-inactivated
virus or purified ligands), IB levels were still significantly
reduced, suggesting that in our system there are viral mechanisms
operating to inhibit IB protein levels. In other reported
systems, IB levels usually return to unstimulated levels within
1 h (39). There were no changes detected in IBß levels under
any of the conditions used in our experiments. It is possible that
there might be changes in IBß levels at later times of infection
or that IBß could be acting as a chaperone for NF-B, protecting
it from IB, as Suyang et al. have shown for some forms of
IBß (63). These results thus suggest that the virus is acting like
an acute stimuli (39, 63, 64), at least under our laboratory
conditions.
Lastly, we showed that there was a rapid increase in p38 phosphorylation (within 15 min), which has been well documented to be correlated to p38 function (65). Furthermore, we showed that viral-mediated signaling (at least IL-1ß production) was dependent on p38 activity as a specific inhibitor of p38 (SB203580) nearly completely blocked IL-1ß message expression (>95% decrease) in all the doses we tested (1–50 µM). These findings thus begin to map the signaling events associated with viral glycoprotein-cellular receptor engagement in monocytes. Excitingly, this event appears to be unique to monocytes because in permissively infected human fibroblasts p38 activity has no function at IE or early times after infection (15 min to 8 h postinfection; (41)), suggesting that in fibroblasts the signaling cascade through these same glycoproteins (gB and gH) diverges following viral binding. These differences may point to the unique and important biological signaling consequences of viral infection in nonpermissive vs permissive cell types. Our on going studies are now addressing these potential differences in more detail.
It is important to point out that it is unlikely in any of the cases presented above that the monocyte activation seen was due to some mitogenic factor in the viral supernatant or in the soluble activating agents (gB or the gH anti-idiotypic Ab) because 1) in the viral binding studies we used density gradient-purified virus; 2) in the gB-mediated studies we used a control protein lysate isolated in the same manner as the purified gB; and 3) in the gH-mediated studies we used the appropriate control Abs. In addition, the Ab blocking studies from these various experiments supported this fact. If a soluble mitogenic factor was copurified with the virus or the gB, its effect would not have been specifically inhibited by the anti-HCMV Abs. The UV-inactivated virus studies, which also resulted in activated gene expression and transcription factors, further supports these points because the studies are similar to the work by Boldogh et al. in which they showed that density-purified virus and inactivated virus had identical effects on protooncogene mRNA levels (31).
In conclusion, we showed that there is a bona fide signal transduction
event that takes place following HCMV binding to monocytes through the
interaction of the surface glycoproteins, gB and gH, on the viral
membrane. We have documented a rapid increase in the levels of nuclear
NF-B DNA binding following the incubation of HCMV with monocytes
that is regulated by the degradation of IB. This transcription
factor induction is then probably responsible for the observed increase
in monocyte mediators (IL-1ß, A20, p105/p50, and IB) and then
ultimately in the increased cytokine production and secretion (as shown
for IL-1ß). This dysregulation of monocyte function by HCMV would
have serious consequences for the infected host because at the very
least an increased cytokine production would profoundly affect the way
cells behave in their natural environment. This aberrant HCMV-mediated
activation of monocytes could be some of the molecular events behind
HCMV’s link to chronic inflammatory diseases such as coronary
restenosis, atherosclerosis, arthritis, and other HCMV-associated
diseases.
|
Acknowledgments |
|---|
Id and control
anti-sera. We also thank S.-M. Huong for her valuable
technical assistance and R. Johnson, Dr. R. Scott,
Dr. L. Su, and B. Faith for their critical review of the
manuscript and helpful discussions. Lastly, we thank Drs.
H.-Y. Fu and Jennifer Webster-Cyriaque for drawing blood and
our valuable blood donors for their invaluable contributions. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Andrew D. Yurochko, Rm. 32–026 Lineberger Comprehensive Cancer Center, University of North Carolina, CB 7295, Chapel Hill, NC 27599-7295. E-mail address:
3 Abbreviations used in this paper: HCMV, human CMV; IE, immediate-early; MIEP, major immediate-early promoter; MAPK, mitogen-activated protein kinase; M.O.I., multiplicity of infection; EMSA, electrophoretic mobility shift assay; NAD, nonadherent; ADH, adherent; Id, anti-idiotypic; PSA, prostate-specific Ag; PSA, anti-prostate-specific Ag; ATF, activation transcription factor; CREB, cAMP-response-element-binding protein; T0', time zero.
Received for publication September 24, 1998. Accepted for publication January 19, 1999.
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J. A. Fairley, J. Baillie, M. Bain, and J. H. Sinclair Human cytomegalovirus infection inhibits epidermal growth factor (EGF) signalling by targeting EGF receptors J. Gen. Virol., November 1, 2002; 83(11): 2803 - 2810. [Abstract] [Full Text] [PDF]
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B. H. Song, G. C. Lee, M. S. Moon, Y. H. Cho, and C. H. Lee Human cytomegalovirus binding to heparan sulfate proteoglycans on the cell surface and/or entry stimulates the expression of human leukocyte antigen class I J. Gen. Virol., October 1, 2001; 82(10): 2405 - 2413. [Abstract] [Full Text] [PDF]
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S.-H. Chiou, J.-H. Liu, W.-M. Hsu, S. S.-L. Chen, S.-Y. Chang, L.-J. Juan, J.-C. Lin, Y.-T. Yang, W.-W. Wong, C.-Y. Liu, et al. Up-Regulation of Fas Ligand Expression by Human Cytomegalovirus Immediate-Early Gene Product 2: A Novel Mechanism in Cytomegalovirus-Induced Apoptosis in Human Retina J. Immunol., October 1, 2001; 167(7): 4098 - 4103. [Abstract] [Full Text] [PDF]
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J. Cinatl Jr, S. Margraf, J.-U. Vogel, M. Scholz, J. Cinatl, and H. W. Doerr Human Cytomegalovirus Circumvents NF-{kappa}B Dependence in Retinal Pigment Epithelial Cells J. Immunol., August 15, 2001; 167(4): 1900 - 1908. [Abstract] [Full Text] [PDF]
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R. A. Johnson, X. Wang, X.-L. Ma, S.-M. Huong, and E.-S. Huang Human Cytomegalovirus Up-Regulates the Phosphatidylinositol 3-Kinase (PI3-K) Pathway: Inhibition of PI3-K Activity Inhibits Viral Replication and Virus-Induced Signaling J. Virol., July 1, 2001; 75(13): 6022 - 6032. [Abstract] [Full Text] [PDF]
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 K. A. Simmen, J. Singh, B. G. M. Luukkonen, M. Lopper, A. Bittner, N. E. Miller, M. R. Jackson, T. Compton, and K. Fruh Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B PNAS, May 30, 2001; (2001) 121177598. [Abstract] [Full Text] [PDF]
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Z. Tabi, M. Moutaftsi, and L. K. Borysiewicz Human Cytomegalovirus pp65- and Immediate Early 1 Antigen-Specific HLA Class I-Restricted Cytotoxic T Cell Responses Induced by Cross-Presentation of Viral Antigens J. Immunol., May 1, 2001; 166(9): 5695 - 5703. [Abstract] [Full Text] [PDF]
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 T. H. Mogensen and S. R. Paludan Molecular Pathways in Virus-Induced Cytokine Production Microbiol. Mol. Biol. Rev., March 1, 2001; 65(1): 131 - 150. [Abstract] [Full Text] [PDF]
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R. A. Johnson, S.-M. Huong, and E.-S. Huang Activation of the Mitogen-Activated Protein Kinase p38 by Human Cytomegalovirus Infection through Two Distinct Pathways: a Novel Mechanism for Activation of p38 J. Virol., February 1, 2000; 74(3): 1158 - 1167. [Abstract] [Full Text]
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K. A. Simmen, J. Singh, B. G. M. Luukkonen, M. Lopper, A. Bittner, N. E. Miller, M. R. Jackson, T. Compton, and K. Fruh Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B PNAS, June 19, 2001; 98(13): 7140 - 7145. [Abstract] [Full Text] [PDF]
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 M. K. Froberg, A. Adams, N. Seacotte, J. Parker-Thornburg, and P. Kolattukudy Cytomegalovirus Infection Accelerates Inflammation in Vascular Tissue Overexpressing Monocyte Chemoattractant Protein-1 Circ. Res., December 7, 2001; 89(12): 1224 - 1230. [Abstract] [Full Text] [PDF]
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