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B activation and IL-1ß synthesis in human monocytes-macrophages involving PKC and PI3-K
Laboratory of Immunovirology, Department of Microbiology and Immunology, and Pediatric Research Center, University of Montreal; Ste. Justine Hospital, Montreal, Quebec, Canada H3T 1C5
1Correspondence: Laboratory of Immunovirology, Ste-Justine Hospital, 3175 Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5. E-mail: svanasve{at}justine.umontreal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B family of
transcription factors.—D’Addario, M., Ahmad, A., Xu, J. W.,
Menezes, J. Epstein-Barr virus envelope glycoprotein gp350 induces
NF-B activation and IL-1ß synthesis in human monocytes-macrophages
involving PKC and PI3-K.
Key Words: EBV gp350 • interleukin 1ß • signal transduction • transcription factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Primary infections with EBV occur via epithelial
cells, which subsequently release virus and lead to infection of B
lymphocytes. EBV binds to and infects cells through complement receptor
type II (CR2 or CD21), which is the natural ligand for the C3d fragment
of the third component of complement (2
3
4
5)
. Glycoprotein
(gp)350 is the major envelope glycoprotein of EBV; it specifically
binds to CR2/CD21 and initiates the viral infection process (5
, 6)
. Apart from initiating this process, gp350 is the major
target protein for EBV-specific humoral and cellular immune responses;
many anti-EBV subunit vaccines currently under human trials are based
on this glycoprotein (reviewed in refs 7
, 8
).
CD21 belongs to a family of proteins containing short consensus repeats
(SCRs), a structural module found in many other proteins associated
with inflammation, tissue repair, and immune responses (reviewed in
refs 2
, 9
). Although originally described on B-lymphocytes, where it
represents an important component of the B cell antigen receptor
signaling complex, CD21/CR2 has also been found on follicular dendritic
and endothelial cells, thymocytes, T cells, epithelial cells, and
monocytes (4
, 10
11
12
13
14
15)
. Thus, EBV may infect and/or
interact with cells of these diverse lineages and modulate their
physiological activities.
Earlier studies from this laboratory have shown that EBV modulates the
synthesis of proinflammatory cytokines interleukin 6 (IL-6), IL-1ß,
and tumor necrosis factor 
(TNF-) from human peripheral blood
mononuclear (PBMC) and monocytic tumor cells (12
, 16
, 17)
.
These effects of EBV on cytokine synthesis in human cells were
dependent on the binding of the viral particles to the EBV receptors on
the target cells, suggesting the involvement of gp350 in this process.
More recently, Tanner et al. (18)
reported the induction
of IL-6 synthesis via protein kinase C (PKC) in human B cells. These
studies indicate that gp350 can interact with CR2/CD21-bearing human
cells and modulate the synthesis of cytokines in them. The effects of
the potential interaction of gp350 with human monocyte-macrophages have
not been investigated, however. These cell types play a pivotal role in
the induction of immune and inflammatory responses, represent an
important source of proinflammatory cytokines in the body, and express
low levels of CR2/CD21 on their surface (12, 14; reviewed in ref 19
).
In this report we have addressed this issue and investigated the
effects of purified recombinant gp350 (rgp350) on the synthesis of
IL-1ß in human plastic-adherent monocyte-macrophages (AMM). IL-1ß
is a potent multifunctional proinflammatory cytokine whose activities
affect almost all other cell types (reviewed in ref 20
). Apart from
playing a crucial role in the regulation of immune and inflammatory
responses, IL-1ß can traverse the blood–brain barrier and therefore
can also affect neurological functions (20)
. Our results
indicate that synthesis of this important cytokine is up-regulated by
rgp350 through pathways that involve enzymatic activities of PKC and
phosphatidylinositol 3,4,5 triphosphate kinase (PI3-K) and activation
of the NF-B family of transcription factors. To our knowledge, this
is the first report describing the immunobiological consequences of the
direct interactions between purified rgp350 and human plastic-adherent
blood monocyte-macrophages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell treatments and preparation of samples
To see the effect of rgp350 on the synthesis of IL-1ß in AMM,
the glycoprotein was added to the (1 ml) cultures of AMM (2 x
105 cells/ml) at a final concentration of 100
ng/ml. This concentration was found optimal in pilot experiments to
induce increased synthesis of this cytokine in PBMC (data not shown).
The cultures were incubated at 37°C, in a 5%
CO2-containing humidified atmosphere for 36 h. Results from pilot experiments indicated that this was the time
point at which the IL-1ß synthesis reached maximum levels in PBMC
when induced with rgp350 or UV-irradiated EBV (UV-EBV). After 36 h
the culture supernatants were collected, centrifuged at 1200 x
g for 15 min at 4°C to remove cells and cellular debris,
and stored at -80°C until used to determine IL-1ß. The cells were
washed with ice-cold phosphate buffered saline (PBS, pH 7.2) and
processed for preparation of cytosolic fractions as described
(21)
. Briefly, the cell pellet was swollen in five packed
cell volumes of a hypotonic buffer containing 10 mM Tris (pH 8.2), 27
mM DMSO, 5 mM PMSF, 10 mM HEPES (pH 8.2), 0.75 mM spermidine, 1 mM DTT,
0.1 mM EDTA, 0.1 mM EGTA, and protease inhibitors as described earlier
(21)
. The cells were lysed in a Dounce homogenizer (20–25
strokes) and the lysates were centrifuged at 45,000 rpm for 30 min at
4°C in a Ti-45 rotor (Beckman Ultracentrifuge, Fullerton, Calif.).
The supernatants were collected, their protein concentration was
determined by using the bicinchoninic acid (BCA) reagent kit (Pierce,
Rockford, Ill.), and aliquots were stored at -80°C until examined.
IL-1ß concentrations were determined in 5 µg proteins of these
cytosolic preparations.
As a positive control for the induction of synthesis of IL-1ß, we treated AMM with TPA (12,0-tetradecanoyl phorbol 13-acetate, 25 ng/ml, Sigma, St. Louis, Mo.). In some experiments rgp350 was pretreated with monoclonal antibodies (mAbs) before adding to the cell cultures; this was done by incubating 100 ng of rgp350 with 5 µg of the antibody at room temperature for 15 min.
To see the effect of CR2-specific mAb OKB7 on the induction of IL-1ß synthesis, this mAb was added to the cell cultures at a final concentration of 10 µg/ml. The cell cultures were kept at room temperature for 30 min before being treated with rgp350.
The cells were also treated with EBV (infectious or noninfectious; see below). For this purpose, the cell pellets were incubated with 100 µl of the virus preparation, vortexed gently, and incubated at 37°C for 1 h. After this, the cells were washed twice with the culture medium, resuspended at 2 x 105 cells/ml concentration, and incubated at 37°C for 36 h.
Unless stated otherwise, all treatments involved 2 x 105 cells in 1 ml volume of the culture medium and incubation for 36 h to determine IL-1ß in the culture supernatants and cytosolic preparations or 6 h for quantitation of IL-1ß mRNA in the cells.
Reagents and antibodies
To determine which signaling pathways were involved in the
induction of IL-1ß by rgp350, the cells were stimulated with this
glycoprotein in the presence of specific inhibitors of various signal
transduction pathway enzymes. The inhibitors used were tyrphostin
AG1478 (an inhibitor of protein tyrosine kinases, epidermal growth
factor receptor, and platelet-derived growth factor receptor, 100
µM), bisindoylmaleimide (a specific inhibitor of PKC, 5 µM),
LY294002 (a specific inhibitor of PI3-K, 40 µM), staurosporine (a
broad spectrum inhibitor of protein kinases, 50 nm), and
MDL-12,330A-HCl (an irreversible inhibitor of adenyl cyclase, 1 mM).
These inhibitors were all purchased from Calbiochem/InterScience
(Markham, Ontario). The concentrations of the inhibitors used are shown
in parentheses and are those recommended by the manufacturer.
Anti-CR2 mAb OKB7 was obtained from Ortho Diagnostic Systems (Raritan, N.J.), which neutralized the binding of rgp350 to target cells (Raji) at 5 µg/ml. Anti-gp350 mAb 2L10, which does not block rgp350 binding to CR2, was a gift from Dr. G. Pearson (Georgetown University, Washington, D.C.); anti-gp350 mAb 72A1, which blocks binding of gp350 to CR2, was kindly provided by Dr. J. Gosselin (Laval University, Quebec, Canada).
Specific monoclonal antibodies for p50, p65, and c-Fos were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.); each was used in electrophoretic mobility shift assays (EMSA) at 1 µg per reaction.
The cell membrane-permeable synthetic peptide SN50 and its control
SN50M were obtained from Calbiochem (San Diego, Calif.). This inhibitor
peptide has been shown to block the nuclear translocation of p50 p65
and c-Rel in human monocytic cells, whereas the control peptide has no
such effect (22)
. Incubation of both SN50 and its control
with AMM cells for up to 5 days did not result in any significant
toxicity.
EBV and rgp350 preparation
EBV was prepared as described previously (23)
.
Briefly, the transforming strain (B95–8) of EBV (24)
was
obtained from cell-free supernatants of B95–8 cell cultures.
Supernatants from 1-wk-old cultures of these cells were filtered
through 0.45 µM filters (Nalge Labware, Corning, New York) and
centrifuged at 45,000 x g for 90 min at 4°C. The
viral pellets were resuspended in PBS to yield 500x concentration of
virus as compared to the culture supernatants. The virus preparation
was titrated by the induction of nuclear antigen (EBNA) in BJAB cells
as described (23)
. The viral preparation used for these
studies contained 2 x 105 EBNA-inducing
units/ml. UV-inactivated virus was obtained by irradiating EBV for 60
min at 265 nm. After UV inactivation, it contained less than 10
EBNA-inducing units/ml.
The rgp350 preparation used was a gift from Dr. Andrew Morgan
(University of Bristol, U.K.). It was produced in a mouse fibroblast
cell line line (C127) after transfection with a bovine papilloma
virus-based expression system (25)
. The gp350 produced,
which lacked the membrane anchor region, was purified from the culture
medium and further clarified using Sephacryl 5300HR and gelatin agarose
(25)
. When examined on a silver stain gel, the protein
gave bands of the expected molecular weight (data not shown) and its
immunological activity was similar to the native EBV-gp350
(25)
. The endotoxin content of the EBV and gp350
preparations was determined and found to be less than 25 pg/ml
according to a Limulus amebocyte lysate-based endotoxin detection kit
(ICN Immunochemicals, Montreal, Canada).
Assay for cytokine concentration
Concentrations of IL-1ß in the cell-free culture supernatants
(secreted from) and in the cytosolic preparation (cytosolic or
cell-associated form) were determined using a commercial ELISA Kit (R&D
Systems, Minneapolis, Minn.) according to the manufacturer’s
instructions. The lowest limit of detection of IL-1ß by this kit was
10 pg/ml and measured the ‘free’ forms of the cytokine.
RNA isolation
RNA isolation was performed using a modified guanidium
thiocyanate procedure (26)
. Briefly, cells were collected
by centrifugation (1200 x g for 10 min), rinsed in PBS
(75 mM NaCl, 2 mM KCl, 8 mM
NaH2PO4), and resuspended
in 1 ml of solution D (4 M guanidium thiocyanate, 25 mM Na citrate, pH
7.0, 0.5% sarcosyl, and 100 mM ß-mercaptoethanol). The cells were
vortexed, placed on ice for 15 min, then centrifuged in an Eppendorf
Microfuge for 20 min at 4°C at 1400 x g after adding
an equal volume of phenol/water, 1/10th volume of chloroform-isoamyl
alcohol, and 1/20th volume of 0.5 M Na acetate (pH 4.0).
Reverse transcription (RT) and polymerase chain reaction (PCR)
analysis
RT was performed on total RNA (1 µg) using 5 U of Moloney
murine leukemia virus reverse transcriptase (Life Technologies,
Inc.-BRL) and 10 pmol of random primers. The mixture was heat-denatured
for 5 min at 85°C. Total reaction volume was 20 µl in a buffer
containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM DTT, and 3 mM
MgCl2. PCR assays were performed in a total
volume of 50 µl using all the RT product in a PCR buffer containing
100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM
MgCl2, 100 µg/ml bovine serum albumin, and 2
µM of each of the four deoxynucleoside triphosphates (Pharmacia), 10
pmol primer A (forward primer), 10 pmol primer B (reverse primer), and
1.0 U Taq DNA polymerase (Promega, Madison, Wis.). The PCR
reactions involved an initial incubation at 95°C for 5 min and then
annealing at 55°C for 1 min, extension at 72°C for 1 min, and
denaturation at 95°C for 1.5 min. Thirty cycles of amplification were
used. All PCR experiments included one control tube with no reverse
transcription step.
PCR-amplified products were resolved in a 1.0% TBE agarose gel,
transferred to nylon membranes, and validated by probing with
32P-end-labeled oligonucleotide probes, which
were used for PCR as described (27)
.
Quantification of the PCR products was performed using the Image-Quant
PhosphorImager (Molecular Dynamics Technologies, Sunnyvale, Calif.) and
normalized with a PCR-amplified housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sequences of the
oligonucleotides used in the RT-PCR analysis were as described
(16)
: IL-1ß, forward primer 5'-ATGGCAGAAGTACCTGAGCTG-3';
reverse primer 5'-TTCCTTGAGGCCCAAGGCCAC-3'; GAPDH forward primer
5'-CCATGGAGAAGGCTGGGG-3'; reverse primer 5'-CAAAGTTGTCATGGAGCC-3'. The
semi-quantitative nature of our RT-PCR protocol, the precautions taken,
and the controls used have all been described (28)
.
Electrophoretic mobility shift assays (EMSA)
To determine the NF-B binding activity in the rgp350-treated
and control cells, EMSA were performed. For this purpose, whole-cell
extracts were prepared as described (21)
. Briefly, cells
were pelleted by centrifugation 4 h after induction by various
agents, washed with ice-cold PBS, and resuspended in 0.5 ml of the
lysis buffer (20 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM
spermidine, 10% glycerol, 10 mM sodium molybdate, 1 mM DTT) containing
protease inhibitors: 0.5 mM PMSF and 1 µg/ml of each of pepstatin,
leupeptin, and aprotinin (all inhibitors from Boehringer Mannheim,
Laval, Québec). Cells were lysed by adding 2 M KCl dropwise to a
final concentration of 0.5 M KCl, gently mixed by rotation at 4°C for
30 min, and centrifuged at 45,000 x g for 1 h at
4°C in a Beckman Ultracentrifuge. The supernatants were diluted to
0.1 M KCl with the lysis buffer and protein concentrations of the
supernatants were determined using BCA protein assay, as described
above.
For EMSA, double-stranded DNA oligonucleotides representing NF-B
sites in IL-1ß promoter (-297 to -288, 5'-GGGAAAATCC-3',
IL-1ß-B), and a mutant version of the IL-1ß-B
5'-ACTAAATTCC-3', which lacks NF-B binding ability, were used. A
double-stranded DNA oligonucleotide corresponding to the c-AMP response
element (CRE) was also used in some assays; all these oligonucleotides
have been described earlier (21
, 27
, 29
, 30)
. Five
micrograms of the protein from whole-cell extracts were preincubated
with 5 µg of poly (dI:dC) for 10 min at 4°C for reducing
nonspecific binding to the oligonucleotides, then 20 ng of the
32P-end-labeled oligonucleotides was added to the
mixture and incubated for 20 min at room temperature. In competition
assays, a 200-fold molar excess of the unlabeled oligonucleotide was
added to the mixture during preincubation. For mobility supershifts,
performed to identify the binding proteins to the oligonucleotides in
EMSA, 1 µg of the p50-, p65-, or c-Fos-specific monoclonal antibodies
was added during the preincubation period. After incubation, the
samples were analyzed on a 6% native Tris-glycine PAGE, migrated at
150 V for 5 h, dried, and exposed to X-ray films for different
lengths of time.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A low-level constitutive production of both forms of this cytokine was
observed in these experiments. The presence of rgp350 in the cultures
caused a marked increase in both the secreted and cytosolic forms of
the cytokine. UV-EBV also stimulated the production of both forms of
IL-1ß. However, in marked contrast to the UV-EBV and rgp350,
infectious EBV lacked this ability; virtually no differences were
observed in the secreted and cytosolic forms of IL-1ß between
infectious EBV-treated and untreated control AMM cultures. Pretreatment
of rgp350 with gp350-specific 72A1, but not with 2L10 mAbs, markedly
inhibited this enhanced production of both forms of this cytokine (Fig. 1
; compare lanes 3, 5, and 6). It is noteworthy that mAb 72A1 blocks
binding of gp350 to CR2/CD21 whereas mAb 2L10 has no significant effect
on this binding (6
, 31)
. These results suggest that the
binding of rgp350 to the EBV receptor CR2/CD21 on AMM was the specific
event that caused the stimulation of IL-1ß production (also see
below). The cytosolic IL-1ß concentration was always higher
(~twofold) than its secreted form in AMM whether or not these cells
were induced.
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Effect of CR2-specific mAb OKB7 on rgp350-mediated stimulation of
IL-1ß production
The results reported above with 2L10- or 72A1-pretreated rgp350
suggest that the binding of this glycoprotein to CR2/CD21 stimulated
the production of IL-1ß. To confirm that this binding was necessary
for the stimulated production of IL-1ß, we pretreated 2 x
105 AMM by adding CR2/CD21-specific mAb OKB7 to
the culture medium (10 µg/ml final concentration) 30 min before
addition of rgp350. This mAb blocks the binding of gp350 and EBV to
CR2/CD21 on target cells (5)
. As shown in Fig. 2
, pretreatment of cells with OKB7 markedly inhibited the rgp350-induced
synthesis of the secreted (~50% reduction) and cytosolic (~75%
reduction) forms of IL-1ß. The treatment of AMM with OKB7 alone did
not have a significant effect on the constitutive synthesis of the two
forms of this cytokine. These results clearly indicate that the
rgp350-induced stimulation of IL-1ß production in AMM is dependent on
the specific binding of this glycoprotein to the EBV receptor CR2/CD21.
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Effect of rgp350 on IL-1ß mRNA
To examine whether the rgp350-induced production of IL-1ß from
AMM was accompanied by increased steady-state levels of IL-1ß mRNA in
these cells, 2 x 105 cells were incubated
in 1 ml of the culture medium and treated with rgp350 (with or without
prior treatment with OKB7), or rgp350 that was pretreated with 2L10,
72A1, or control antibodies at 37°C as described above. After 6 h of incubation, the cells were collected and processed for
determination of IL-1ß mRNA by the RT-PCR method as described in
Materials and Methods. As shown in Fig. 3A
, pretreatment of cells with OKB7 and pretreatment of rgp350
with 72A1 caused a marked reduction in the induction of IL-1ß mRNA
expression.
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A kinetic study of IL-1ß mRNA induction by rgp350 was undertaken.
Figure 3B
shows the relative levels of IL-1ß mRNA in
rgp350-treated AMM (100 ng of rgp350 per 2 x
105 AMM/ml of the medium) at different time
points after the treatment. IL-1ß mRNA was induced as early as 1 h after adding rgp350 to the cultures and reached a peak level at 4–6
h post-treatment. Since UV-EBV behaved essentially like rgp350 in
inducing the synthesis of IL-1ß in AMM, we repeated this kinetic
experiment using UV-EBV. As shown in Fig. 3C
, UV-EBV also
induced IL-1ß mRNA in AMM by 1 h; however, the levels tended to
decline earlier.
Although AMM constitutively express low basal levels of secreted and cytosolic forms of IL-1ß, in our hands no signal for IL-1ß mRNA was visible in untreated cells in the two autoradiograms shown here. This was simply due to shorter exposure times of these blots to X-ray films, since upon prolonged exposure IL-1ß messages were detected in untreated cells. These exposures, however, caused over-darkening and intermingling of signals from treated cells (data not shown). These results clearly demonstrate that treatment of human AMM with rgp350 or UV-EBV causes a rapid increase in the steady-state level of IL-1ß mRNA; furthermore, this increase in IL-1ß is induced by specific binding of gp350 to CR2/CD21 on these cells.
The signal-transduction pathways involved in rgp350-mediated
induction of IL-1ß production
It has been suggested that because of its short cytoplasmic tail,
CR2/CD21 is unable to transduce signals intracellularly
(32)
. However, the data presented above clearly indicate
that upon binding to rgp350 or UV-EBV, CR2/CD21 can transduce signals
that result in the induction of IL-1ß synthesis. To determine the
nature of the signal transduction pathway(s) used by CD21/CR2 in this
IL-1ß induction, we treated AMM with rgp350 (100 ng/ml) with or
without the presence of reagents that inhibit activities of various
kinases. These reagents included specific and nonspecific inhibitors
for adenyl cyclase, PKC, PI3-K, and protein tyrosine kinases. These
inhibitors and the concentrations used are provided in Materials and
Methods. The cells were incubated at 37°C for 6 h and the
quantity of IL-1ß mRNA was determined by RT-PCR, as described above.
The results are depicted in Fig. 4
and Table 1
and demonstrate that the induction of IL-1ß
mRNA was markedly reduced when cells were stimulated with rgp350 in the
presence of specific inhibitors for PKC (80% reduction, lane 6 in Fig. 4
) and PI3-K (50% reduction, lane 7 in Fig. 4
). Even when used at
higher concentrations (e.g., up to 50 nM for staurosporin), only PKC
and PI3-K inhibitors blocked the induction of IL-1ß mRNA; other
inhibitors had no significant effect (data not shown).
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Activation of NF-B in rgp350-treated cells
A rapid induction of IL-1ß mRNA in AMM after treatment with
rgp350 suggested the involvement of rapidly inducible transcription
factors (e.g., NF-B) in this process. Furthermore, IL-1ß is a
proinflammatory cytokine and constitutive, chronic activation of
NF-B in inflammatory conditions is well known (reviewed in refs 33
34
35
). These considerations and the fact that three NF-B binding
sites have been demonstrated in the promoter region of IL-1ß gene
(28
, 29
, 36)
prompted us to investigate whether rgp350
treatment induces NF-B binding activity in human AMM. Therefore, we
conducted EMSA assays using double-stranded DNA oligonucleotides
representing NF-B binding sites in the IL-1ß as well as a mutant
site (detailed in Materials and Methods). As shown in Fig. 5A
, whole-cell extracts from untreated AMM showed constitutive
NF-B binding activity (consistent with the low-level synthesis of
this cytokine in these cells) that was markedly increased (~8.0-fold)
on treatment with rgp350. The specificity of the NF-B binding in
EMSA was determined by cold competition with the mutant site and with
unlabeled IL-1ß-B oligonucleotide. Addition of the cold
IL-1ß-B to the assays abrogated binding to the oligonucleotide
probe, whereas competition with the mutant IL-1ß-B did not cause
this reduction (Fig. 5A
, lanes 5, Fig. 6
). In addition to NF-B, the IL-1ß promoter region also contains
multiple CREB (cyclic AMP-responsive element binding protein) sites.
Our results using gp350 or EBV showed no enhanced CREB binding (data
not shown).
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To
confirmthat the complexes bound to the IL-1ß-B oligonucleotides from the
whole-cell extracts of rgp350-treated AMM contained authentic
transcription factors belonging to the NF-B/Rel family, we added
p50-, p65,- or c-Fos-specific mAbs (1 µg/reaction) during
preincubation with poly (dI:dC) and performed the EMSA. The p65 (RelA)
and p50 are classical prototypic members of the NF-B/Rel family
whereas c-Fos is a component of another inducible transcription factor,
activation protein-1 (AP-1; refs 33
, 35
). As shown in Fig. 5A
, the IL-1ß-B bound complexes were supershifted when
anti-p50 or anti-p65 antibodies were added but not when
anti-c-Fos-antibodies were added. The relative strength of the
supershift was considerably greater with anti-p50 antibody than with
anti-p65 antibody. These results suggest that treatment of AMM with
rgp350 induces the activation of p50 and p65 in these cells.
Finally, we determined whether gp350-specific monoclonal antibodies and
PI3-K and PKC inhibitors that inhibit rgp350-induced activation of
IL-1ß gene also inhibit rgp350-mediated NF-B activation in human
monocyte-macrophages. We pretreated these cell cultures with these
regents, treated them with rgp350, and determined NF-B activation by
EMSA (see Materials and Methods). As shown in Fig. 5B
, 72A1
mAb and specific inhibitors of PKC and PI3-K significantly inhibited
this activation in these cells, which is consistent with our data on
the inhibition of IL-1ß gene activation by these reagents.
Inhibition of rgp350-mediated NF-B activation inhibits the
activation of the IL-1ß gene
To find out whether rgp350-mediated activation of NF-B is
essential for the activation of the IL-1ß gene, the AMM were
incubated with this glycoprotein in the presence of a cell-permeable
synthetic peptide SN50 that has been shown to prevent the nuclear
translocation of at least three members of the NF-B family (p50,
p65, and c-Rel) in human monocytic cells (22)
. This is
tantamount to inhibition of the activation of these factors, since
without their translocation to the cell nucleus they cannot bind to the
target DNA sequences and mediate their effects. As shown in Fig. 6
, the
inhibitor peptide not only significantly reduced the production of
IL-1ß in the culture supernatants, but also decreased the
steady-state levels of IL-1ß mRNA as compared to the rgp350-treated
cells. The control peptide had no significant effect on IL-1ß gene
activation in rgp350-treated cells, suggesting that the activation of
NF-B in rgp350-treated cells was a prerequisite for IL-1ß gene
expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 16
, 17)
. Using unfractionated PBMC, we had shown that
EBV remarkably down-regulated TNF- production in these cells whereas
its effects on IL-1ß production were slightly stimulatory (12
, 16
, 17)
.
The differences in the results of these and our previous studies
(12
, 16
, 17)
may be explained by the fact that earlier we
had used whole, unfractionated PBMC whereas in the present study we
used purified monocyte-macrophage cultures. It is quite possible that
one or more subpopulations of PBMC may be responding to EBV in a
different way than monocyte-macrophages with respect to the activation
of IL-1ß gene. Gp350 has recently been shown to induce the production
of IL-6 in human B cells (18)
; however, to our knowledge
this the first report implicating this glycoprotein in the induction of
proinflammatory cytokines from human monocyte-macrophages. In addition,
whereas gp350 strongly induces IL-1ß mRNA and protein, EBV appears to
slightly increase IL-1ß RNA production while not enhancing protein
expression; this suggests the existence of some form of
post-transcriptional regulation. We and others earlier demonstrated
that human monocytic cells express CR2 (12
, 14)
, although
these cells are not known as being EBV targets in vivo. It
is conceivable that other CR2-expressing cell types (e.g., T and
follicular dendritic cells) may also be affected by the
immune-modulating properties of EBV and gp350. We have incubated
infectious EBV with AMM cells for up to 7 days, but were not able to
show EBV RNA or protein from these cells, suggesting that this virus
may simply interact with their cell membrane receptors without
undergoing replication (unpublished observations). It has been shown
that EBV interacts with CR2/CD21-expressing thymocytes and interferes
with the thymic selection process by inducing IL-2 from them
(37)
. Furthermore, we recently demonstrated that EBV
causes the release of TGF-ß from human platelets by binding to its
receptor on these blood elements (38)
.
The binding of gp350 to CR2/CD21 on target cells is the first step in
the EBV infection process and the production of cytokines like IL-1ß
(which could induce a strong anti-viral immune and inflammatory
response in the infected host) may not be desirable from the virus
point of view. Thus, EBV has devised strategies to block this response.
These strategies may be in the form of a component(s) of the viral
particle that suppresses this response but is somehow inactivated in
the UV-irradiated virus. In an alternate and more likely (but not
mutually exclusive with the preceding) scenario, infectious EBV may
induce the expression of one or more viral and/or cellular genes
immediately after infection, which prevents the production of
gp350-mediated cytokines. One obvious viral candidate gene for this
effect is the viral homologue of human IL-10 gene (BCRF1 or vIL-10;
1
, 8
), which is comparable to hIL-10 in its capacity to
inhibit cytokine induction (39
, 40)
. We are currently
investigating the role of vIL-10 in the down-regulation of
gp350-induced IL-1ß production.
The present results demonstrate that rgp350 induces the activation of
NF-B or the Rel family of transcription factors in a PI3-K- and
PKC-dependent manner. These transcription factors, which occur as
inactive homo and/or heterodimers in the cytoplasm due to their binding
with inhibitory proteins IB, can be rapidly activated by a wide
variety of stimuli (LPS, PMA, IL-1ß, TNF-, etc.; reviewed in refs 33
, 35
). These stimuli induce phosphorylation at specific
serine/threonine sites of IB, which then become polyubiquinated and
degraded via proteasomes. The degradation of classical IB unmasks
the nuclear localization signals on NF-B dimers, which then migrate
to nucleus and bind to specific response elements—NF-B binding
sites. These sites exist in the promoter regions of numerous genes
involved in cellular growth, differentiation, and inflammatory and
immune responses. Three such sites have been found in the regulatory
region of the IL-1ß gene, and NF-B activation is known to induce
IL-1ß gene expression (41
, 42)
. These sites also exist
in viral promoters, e.g., HIV-I LTR. IL-1ß enhances HIV-1 replication
through activation of NF-B, which bind to NF-B binding sites in
the viral LTR (reviewed in ref 33
). Activation of NF-B may also be
needed for a successful EBV infection. It is noteworthy that EBV
infects resting B cells, which have little or no constitutively
activated NF-B. Sugano et al. (43)
showed that EBV
activates this family of transcription factors via CR2/CD21 in
tonsillar B cells. This activation is PKC dependent and is needed to
drive transcription from the Wp promoter. Wp is the initial EBV latent
gene promoter located in the major long terminal repeat (BH1W; ref 1
);
EBNA2 and EBNA leader proteins are initially transcribed from this
promoter. Our results indicate that rgp350 can also activate these
transcription factors in human monocyte-macrophages via PKC and PI3-K.
Further work will be needed to know whether this activation involves
induced phosphorylation and degradation of IB, increased production
of the transcription factors, or affects the activities of PP2A or of
the newly discovered IB kinases.
The EBV receptor (CR2) is a 140 kDa type II integral membrane protein
and belongs to the regulators of complement activation gene family
(reviewed in refs 2
, 9
). It is an important member of the B cell
antigen receptor complex and can dramatically augment the immune
response to an antigen if the latter is bound to the natural ligand of
CR2, i.e., C3dg (9)
. It occurs singly as well as in
association with CD19, TAPA-1 (target for anti-proliferation
antibody-1), and CD35 (reviewed in refs 2
, 9
). It uses CD19 and/or
TAPA-1 and transduces signals via phospholipase C and PI3-K. Activation
of B cells causes phosphorylation of CR2, cross-linking of CR2 by
extracellular ligands, including EBV on B cells, and increases their
proliferation. C3d or a 16 amino acid peptide corresponding to the CR2
binding domain of C3d has also been shown to phosphorylate pp105 in
CR2-positive cells (44)
. CR2 can also interact with tumor
suppressor protein p53, a Ca2+ binding protein
p68, and with ribonucleoprotein p120 (45)
. It has recently
been shown that binding of EBV to CD21/CR2 on B-lymphocytes activates
PI-3-K independent of CD19 (46)
; these results, along with
our previous findings demonstrating a lack of CD19, suggest that the
presence of CD21 on AMM cells may be sufficient to transduce signals
through the cell membrane, resulting in cytokine gene activation. Our
results also suggest involvement of the PI-3-K pathway for cellular
activation, in agreement with the results shown in B-lymphocytes
(46)
. Although CR2 occurs on monocytic cells, these cells
are devoid of CD19 (2)
. Our results suggest that despite
the lack of CD19, rgp350-dependent stimulation of CR2 in these cells
activates NF-B in addition to PI-3K and PKC (see below).
A wide variety of extracellular stimuli can cause activation of
NF-B. Chronic inflammatory conditions are usually accompanied by the
constitutive high-level activation of these factors. Viral transforming
proteins, e.g., Tax of HTLV-1 and LMP-1 of EBV activate NF-B
(47
, 48)
. It has been shown that activated NF-B
prevents cellular apoptosis (49
, 50)
. Activation of these
transcription factors by these viral proteins may be important for
their cell transforming ability. The present study shows for the first
time the involvement of a nontransforming envelope glycoprotein of EBV
(i.e., gp350) in the activation of these factors in human
monocyte-macrophages. Hemagglutinin of influenza virus, another virus
that infects quiescent cells, is known to activate these transcription
factors (51)
. Thus, it appears that viruses that infect
quiescent cells have evolved the strategy to activate NF-B or other
transcription factors at the beginning of their infection process.
The data presented here also show that rgp350-induced activation of
NF-B in human monocyte-macrophages is PKC and PI3-K dependent. PKC
is a cytosolic serine/threonine kinase that upon activation is
translocated to the cell membrane (reviewed in ref 52
). It has been
shown that gp350 induces IL-6 production in human B cells via
activation of PKC (18)
. Our results suggest that this
glycoprotein can also activate this kinase in human
monocyte-macrophages. Whether PCK activation in gp350-stimulated AMM
also results in the activation of transcription factors other than
NF-B is not known. Our unpublished data suggest that transcription
factors AP-1, CREB, and STAT-3 are not activated in human
monocyte-macrophages by this glycoprotein.
PI3-K catalyzes the synthesis of second messengers
phosphatidylinositol-3,4 biphosphates and -3, 4, 5 triphosphates
(reviewed in ref 53
). This is an important serine/threonine kinase that
is involved in diverse processes such as transformation, inflammation,
cell growth, etc., and is activated by gp350. One of the most important
efferent functions of P13-K is the activation of cellular AKT, a
homologue of viral oncogene AKT that is known to prevent apoptosis
(53)
. Recently, PI3-K itself has been shown to have
transforming ability (54)
. In keeping with the ability of
gp350 to activate NF-B and PI3-K, it is tempting to speculate a role
for gp350 in apoptosis and cell survival.
Because of the potent and widespread inflammatory effects in the human
body, the production and activities of IL-1ß are tightly regulated at
multiple steps: at transcription, mRNA stability, translation,
processing of the protein, and secretion (reviewed in ref 20
). Our work
shows an increase in the steady-state levels of IL-1ß mRNA in
rgp350-treated cells, which may be due to increased mRNA stability
and/or increased transcription. More important, the increased mRNA
levels result in increased production of IL-1ß protein. gp350 differs
in this respect from certain other biological inducers of
IL-1ß—e.g., C5a, which causes an increase in IL-1ß mRNA but no
increase in IL-1ß protein production (55)
. Further work
is required to determine whether gp350 modulates IL-1ß production at
other steps of its regulatory mechanism.
The rgp350-mediated stimulation of IL-1ß production in human
monocyte-macrophages reported here has implications for EBV biology not
only in terms of virus–cell interactions, but also for its immunology
and vaccinology. Gp350 is a target protein for anti-EBV cellular and
humoral immunity. It is expressed most abundantly on the virion and on
the surfaces of productively infected cells. Gp350-based vaccines have
proved effective in simian models in protecting animals from
EBV-induced fatal lymphomas (56)
. By inducing the
secretion of proinflammatory cytokines, the gp350 itself may be acting
as an adjuvant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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