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Viral Immunology Laboratory, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier de l’Université Laval, Québec, Canada; and
Molecular Inflammation Laboratory, Department of Microbiology & Immunology, The University of Adelaide, Adelaide, South Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1). Neutrophil
function is also known to be modulated by priming with
granulocyte-macrophage colony-stimulating factor (GM-CSF). We have
therefore investigated the modulation of EBV-induced activation of
human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF
before EBV activation enhanced the production of both MIP-1 and
IL-8. The IL-8 produced under these conditions was biologically active
as determined in the calcium mobilization assay. GM-CSF was also found
to increase the ability of EBV to prime neutrophils for increased
leukotriene B4 (LTB4) synthesis. Prior
treatment of GM-CSF with neutralizing Abs inhibited these effects.
GM-CSF also increased the specific binding of FITC-EBV to the
neutrophil surface, as evaluated by fluorocytometry. Local production
of GM-CSF in tissues invaded by EBV could therefore serve to potentiate
a host defense mechanism directed toward the destruction of the
infectious virus via increased production of chemotactic factors. Since
both IL-8 and MIP-1 are reported to be chemoattractants in vitro for
T cells and T and B cells, respectively, the ability of EBV to induce
their production by neutrophils may enhance its ability to infect B and
T lymphocytes via increased recruitment to sites of infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils play an important role in the control of viral infections.
They are rapidly mobilized at sites of invasion in the early phases of
infection and have the capacity to synthesize a variety of proteins
that are involved in effector functions for neutrophils as well as
other cell types (8, 9). The recruitment of neutrophils to diseased
tissue is a critical component of host defense. This process is
controlled by various molecules including adhesion molecules and
chemotactic factors. Among the latter, IL-8, macrophage inflammatory
protein-1 (MIP-1), and LTB4 are of particular
interest with resepect to neutrophil biology (10, 11). IL-8, a CXC
chemokine, is strongly chemotactic for neutrophils (reviewed in 11 . MIP-1, a CC chemokine, exhibits chemoattractant potential for
monocytes and varyious lymphocyte subpopulations (12, 13).
LTB4 is a potent proinflammatory lipid produced by the
dioxygenation of arachidonic acid via the 5-lipoxygenase (5-LO)
pathway. While the immune functions of LTB4 are not yet
fully understood, its chemotactic activity for phagocytes such as
neutrophils, monocytes, and macrophages is believed to represent its
most important biologic function (14). Activated neutrophils are
capable of producing all three of these molecules, giving them the
potential to exert autocrine as well as paracrine effects on leukocyte
recruitment in diseased tissues.
EBV, a prominent member of the Herpetoviridae family, is a
lymphotrophic virus known to cause several pathologies in the
lymphocyte compartment. These include infectious mononucleosis and
immunoblastic lymphoma, as well as Burkitt’s lymphoma and
undifferentiated nasopharyngeal carcinoma (15, 16, 17). However, there is a
growing body of evidence to suggest that the interaction between EBV
and cells of the innate immune system, such as neutrophils and
mononuclear phagocytes, leads to significant activation of these cell
types. In previous studies designed to evaluate the EBV/phagocyte
interactions, we observed that EBV binds to the surface of monocytes,
which leads to activation of IL-6 gene expression and inhibition
of TNF- synthesis (15, 16, 17, 18, 19). More recently, we have
demonstrated that EBV interacts with human neutrophils to modulate the
synthesis of various proteins. Of particular interest is the
observation that interaction between EBV and neutrophils modulates the
expression of IL-1 and -ß and IL-1Ra in such a way as to favor the
production of IL-1Ra (20, 21). We have postulated that this may affect
neutrophil-derived IL-1 synthesis during EBV infection. Impairment of
the biologic functions of neutrophils or cytokine activities may then
alter the immune response, which could in turn promote the spread of
the invading agent.
Several lines of evidence indicate that cytokines may play a
significant role in the pathogenesis associated with EBV. First,
elevated levels of IL-1, IL-2, IL-6 and IFN-
have been detected
in sera from infectious mononucleosis (IM) patients during the acute
phase of infection (22, 23). Second, in situ hybridization of tonsils
from patients suffering from IM showed that IL-1ß, IL-6, and TNF-
expression was strongly enhanced in EBV-infected cells (24, 25).
Finally, in the same tissue samples, high levels of IL-1 and -ß
and IL-8 transcripts were detected in neighboring EBV-negative cells
from interfollicular areas (25). Finally, GM-CSF has been found to
facilitate the spontaneous outgrowth of EBV-infected B cells from IM
patients (26).
All of these studies suggest the involvement of chemokines/cytokines in the control of EBV infections. The present study was initiated, therefore, to evaluate the effect of EBV on the synthesis of chemotactic factors by human neutrophils and its interactions with GM-CSF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ficoll-Paque and Dextran T-500 were from Pharmacia (Dorval, Québec, Canada). HBSS, HEPES buffer, RPMI 1640, and FBS were from Life Technologies (Grand Island, NY). Diff-Quick stain kit was from American Scientific Products (McGaw Park, IL). All reagents used for this study were either pharmaceutical grade or contained <5 pg/ml of endotoxin as determined by the Amebocyte lysate assay (BioWhittaker, Walkersville, MD). Biosynthetic human rGM-CSF was a generous gift from the Genetics Institute (Cambridge, MA). The GM-CSF was stored at a stock solution of 100 nM in PBS containing 0.01% BSA (Sigma). At the highest concentration used, GM-CSF had levels of endotoxin contamination < 0.27 pg/ml.
Virus preparation
Preparations of EBV strain B95-8 were produced as previously
described (20, 27). Briefly, B95-8 (mycoplasma free) was grown in RPMI
1640 medium supplemented with 10% heat-inactivated FBS. When the
viability of the cells was 20% or less, cell-free culture supernatants
were harvested and filtered through a 0.45-mm pore size filter, and the
viral particles were concentrated by ultracentrifugation. The virus
pellet was suspended in 5 mM sodium phosphate (pH 7.5) and purified by
centrifugation on a 10 to 30% (w/v) dextran gradient. Concentrated
viral preparations were resuspended in RPMI 1640, aliquoted, and stored
at -80°C until used. The viral titers were determined as previously
described (26) and adjusted to 1 x 107
transforming units (TFU)/ml. No IL-1, IL-1ß, TNF-, GM-CSF,
IL-8, or MIP-1, as assessed by specific ELISA, was detected in the
EBV preparations used.
Purification of neutrophils
Venous blood from healthy medication-free volunteers was collected under sterile conditions using heparin as anticoagulant. Neutrophils were purified by means of 6% dextran sedimentation followed by standard techniques of Ficoll-Paque gradient and hypotonic lysis of erythrocytes (28). Neutrophils were resuspended in RPMI 1640 supplemented with 10% FBS at a final concentration of 107 cells/ml. Differential counts on neutrophil fractions were conducted by Diff-Quick and nonspecific esterase staining. Final neutrophil preparations used in this study were >98% pure, and only preparations containing <2 x 104 monocytes/107 cells were used. Cell viability exceeded 97% as detected by trypan blue exclusion and lactate dehydrogenase activity.
Enzyme immunoassay for immunoreactive IL-8 and MIP-1
The assessment of IL-8 in supernatants and cell-associated
materials was performed using a commercially available enzyme
immunoassay kit purchased from Biosource International (Camarillo, CA).
This IL-8 assay had a minimal detectable level of 5 pg/ml and did not
cross-react significantly with other known cytokines. MIP-1 levels
were assessed using an ELISA kit purchased from R&D Systems
(Minneapolis, MN). This MIP-1 assay had a minimal detectable level
of 2 pg/ml and did not cross-react significantly with other known
cytokines.
Analysis of 5-LO products
For these experiments, neutrophils were resuspended at 5 x 107/ml in serum-free HBSS containing 10 mM HEPES and 1.6 mM CaCl2. Neutrophils were preincubated with or without 200 pM GM-CSF for 30 min at 37°C, incubated with or without EBV for 30 min at 37°C, and further stimulated with or without 50 nM ionophore A23187 for 10 min at 37°C. The reactions were stopped by the addition of 1 volume of a mixture of methanol/acetonitrile (50/50, V/V) containing 12.5 ng/ml each of prostaglandin B2 (PGB2) and 19-hydroxy-PGB2 as internal standards for HPLC analysis. The denatured reaction mixtures were stored overnight at -20°C. The denatured samples were centrifuged at 2000 x g for 20 min to remove the precipitated materials, and LTB4 levels were analyzed by reverse phase HPLC (RP-HPLC) using an on-line extraction procedure previously described (29).
Calcium mobilization
Intracellular free calcium was monitored using the fluorescent probe fura-2/AM as previously described (30). Briefly, neutrophil suspensions (1 x 107/ml) were incubated with 1 µM fura-2/AM for 30 min at 37°C. The cells were then washed free of the extracellular probe, resuspended at 5 x 106 cells/ml, and allowed to re-equilibrate for 10 min at 37°C. The cells were then transferred to the thermostatted (37°C) cuvette compartment of a fluorometer (SLM Aminco, Rochester, NY), and the fluorescence was monitored (excitation and emission wavelength, 340 and 510 nm, respectively). The internal calcium concentrations were calculated as described by Tsien et al. (31).
Isolation of total RNA and Northern blot analysis
Neutrophils were preincubated with or without GM-CSF for 1
h and further incubated with EBV for 3 h before RNA extraction.
Isolation of total cellular RNA and Northern blots were performed as
previously described (32, 33). Briefly, total RNA was isolated by the
RNAzol method (ID Laboratories, London, Ontario, Canada) according to
the manufacturer’s protocol, and 10 µg of total RNA was loaded on a
1% agarose denaturing gel and size fractionated by electrophoresis.
Transfer onto Hybond-N (Amersham Canada Limited, Oakville, Ontario,
Canada) was performed in 3 h with the VacuGene TM XL vacuum
blotting system used according to the manufacturer’s specifications
(Pharmacia, Piscataway, NJ) and was complete for each lane. After
prehybridization, the membranes were hybridized with random-primed
32P-labeled probes according to the Prime-a-Gene
labeling system (Promega Corp., Madison, WI) in 50% formamide
overnight at 42°C. The membranes were then washed and exposed to
Kodak X-OMAT films (Eastman Kodak, Rochester, NY) with an intensifying
screen at -70°C. The human MIP-1 cDNA was synthesized by PCR with
an antisense primer made against nucleotides 283 to 236 of the
published LD78 sequence and a sense primer comprising nucleotides -19
to 3. The IL-8 probe used in this study was a 244-base pair
PstI/EcoRI cDNA fragment representing the coding
region of the IL-8 cDNA from nucleotides 49 to 293. The glyceraldehyde
3-phosphate dehydrogenase (GAPDH) cDNA was used as internal control to
demonstrate the equal loading of RNA in each lane.
Assay for EBV binding
Purified EBV was labeled with FITC, and the labeled virus (FITC-EBV) was separated from unbound FITC using a sephadex G-25 column as previously described (20). Neutrophils (3 x 106) were pretreated with GM-CSF or diluent control before incubation with FITC-EBV for 30 min in the dark at 4°C with frequent agitation. The cells were then washed three times in cold PBS, resuspended, and fixed in 0.5 ml of paraformaldehyde in PBS. The percentage of positive cells was determined from a sample of 104 cells using an EPICS XL (Coulter, Miami Lakes, FL). The specificity of EBV binding was determined by preincubation of neutrophils with excess unlabeled EBV (15 min at 4°C) before incubation with FITC-EBV.
Statistical analysis
Statistical analyses were performed using Student’s paired (two-tailed) t test, and significance was considered attained at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the effects of GM-CSF on EBV-induced IL-8 synthesis in
neutrophils, neutrophils from six normal individuals were pretreated
with 1 nM GM-CSF for 1 h. These conditions were chosen on the
basis of the results of our previous studies on the effects of GM-CSF
on neutrophil activation (6, 33, 34, 35, 36). The cells were then incubated in
the presence or absence of EBV for 8 h. This incubation time was
chosen from the results of previous studies examining the kinetics of
chemokine synthesis in neutrophils (37). Cell-associated and -secreted
materials were examined for the presence of IL-8 using a specific ELISA
(Table I
). Freshly isolated neutrophils
contained constitutive levels of IL-8 that were not altered by
incubation in medium alone for up to 24 h (data not shown). GM-CSF
or EBV alone were found to be potent activators of IL-8 production by
neutrophils. However, 
92 and 80% of the IL-8 produced in response
to GM-CSF or EBV, respectively, remained cell associated. Pretreatment
of neutrophils with GM-CSF before incubation with EBV synergistically
enhanced the total production of IL-8 compared with that induced by
GM-CSF or EBV alone. Moreover, pretreatment with GM-CSF resulted in a
significantly greater proportion of the newly synthesized IL-8 being
secreted (41% of the IL-8 produced was detected in the secreted
materials).
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by
EBV-stimulated neutrophils
We next evaluated the effect of GM-CSF on EBV-induced MIP-1
production by neutrophils. Neutrophils incubated in medium alone failed
to produce MIP-1 (Table II).
Neutrophils were found to synthesize significant amounts of MIP-1
when incubated with EBV alone during 8 h, whereas GM-CSF alone
exerted only a minor effect on MIP-1 production. In contrast to the
effect observed with IL-8, approximately 96% of the MIP-1 produced
in response to EBV was secreted. Pretreatment of neutrophils with
GM-CSF before stimulation with EBV synergistically increased the
production of MIP-1 compared with that induced by EBV and GM-CSF
alone. In this case, the level of secreted MIP-1 was increased
2.2-fold.
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Since pretreatment of neutrophils with GM-CSF before EBV
stimulation resulted in the release of a relatively large amount of
IL-8, it was considered important to determine whether the secreted
IL-8 was biologically active. Supernatants from nonactivated
neutrophils and from GM-CSF-treated neutrophils incubated with EBV were
collected and tested for their ability to induce the mobilization of
calcium in neutrophils (data not shown). The results of a
representative experiment are shown in Figure 1. Supernatants from cells incubated in
medium alone failed to induce calcium mobilization in neutrophils. In
contrast, the supernatants collected from GM-CSF-treated neutrophils
incubated with EBV induced a rapid mobilization of calcium. This
mobilization of calcium was almost totally abrogated when the
supernatants were preincubated with neutralizing mAbs to IL-8,
suggesting that the mobilization of calcium observed was entirely
mediated by IL-8.
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In previous studies, we have observed that EBV primes neutrophils
for O
generation (20) and for
LTB4 release (Gosselin et al., manuscript in
preparation) in response to a second stimulus. To evaluate the effects
of GM-CSF on this priming by EBV, neutrophils were preincubated with
GM-CSF for 30 min, followed by EBV for a further 30 min before
stimulating the cells with calcium ionophore (A23187, 50 nM) for 5 min.
LTB4 synthesis was then assayed by RP-HPLC. As shown in
Figure 2, neutrophils stimulated with a
suboptimal concentration of A23187, with GM-CSF or EBV alone failed to
produce significant levels of LTB4. Preincubation of
neutrophils with GM-CSF before incubation with either EBV or A23187
resulted in the production of LTB4. Preincubation of
neutrophils with EBV before stimulation with A23187 led to the release
of significantly more LTB4 than observed with either EBV or
A23187 alone, demonstrating the priming effect of EBV on
LTB4 production. Finally, pretreatment with GM-CSF strongly
enhanced the priming effect of EBV on LTB4 production by
neutrophils in response to A23187.
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To evaluate the specificity of the enhancing effects of GM-CSF on
EBV-induced chemotactic factor release in neutrophils, GM-CSF was
preincubated with neutralizing or isotype-matched control Abs before
cellular treatment. As shown in Table III, neutralized GM-CSF had no effect on
IL-8 or MIP-1 synthesis in neutrophils treated with EBV or the
control. These results confirm that the enhancing effects observed are
dependent on GM-CSF.
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mRNA
accumulation in neutrophils
The steady state levels of IL-8 and MIP-1 mRNA in neutrophils
preincubated with or without GM-CSF for 1 h at 37°C, and further
incubated with or without EBV for 3 h, were next investigated.
These conditions were found to be optimal for MIP-1 and IL-8 mRNA
accumulation (data not shown). Figure 3
depicts a representative autoradiograph of three such experiments in
which the same membrane was probed with 32P-labeled
IL-8, MIP-1, and GAPDH cDNA, respectively. Neutrophils incubated in
medium alone failed to express detectable levels of IL-8 or MIP-1.
An increase in the level of IL-8 and MIP-1 mRNA was observed when
neutrophils were incubated in the presence of EBV. GM-CSF alone
increased the steady state mRNA levels of IL-8 but failed to affect
that of MIP-1. Furthermore, treatment of neutrophils with GM-CSF
before incubation with EBV did not modify the levels of IL-8 or
MIP-1 mRNA compared with those obtained in response to either GM-CSF
or EBV alone.
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To determine whether incubation of neutrophils with GM-CSF
increased specific binding of EBV, flow cytometry was performed to
evaluate the level of binding of FITC-labeled EBV to neutrophils.
Neutrophils were preincubated for 1 h at 37°C with 3 nm GM-CSF
or diluent control. The cells were then processed for assessment of
binding of EBV. Representative results are shown in Figure 4. Approximately 35 ± 7%
(n = 4; mean ± SD) of diluent-treated
neutrophils were found to bind FITC-EBV (Fig. 4A).
This increased to 71 ± 5% (n = 4)
following a 1-h preincubation with GM-CSF (Fig. 4B).
Finally, incubation of neutrophil with unlabeled EBV (15 min at 4°C)
before incubation with FITC-EBV effectively eliminated binding of
FITC-EBV to neutrophils (4 ± 4%; n = 4),
demonstrating the specificity of the interaction (Fig. 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induced by EBV and
increases the priming effect induced by EBV on LTB4
production by neutrophils. This demonstrates that GM-CSF both primes
neutrophils for increased direct as well as indirect responses to
EBV.
Pretreatment with GM-CSF had differential effects on the expression of
the chemokines IL-8 and MIP-1. As previously reported (38), GM-CSF
induced significant expression of IL-8 but exerted a minor effect on
MIP-1 expression, indicating differential regulation of these two
genes by GM-CSF. Despite this fact, pretreatment with GM-CSF increased
the production of both IL-8 and MIP-1 induced by subsequent exposure
to EBV by approximately two- to three-fold in both cases. A further
difference with respect to production of the two chemokines lies at the
level of secretion. The majority of IL-8 produced in response to either
GM-CSF or EBV alone remained cell-associated, whereas the combination
of the two agonists resulted in significantly more of the IL-8 being
secreted. This is in contrast to the regulation of MIP-1, in which
the majority of the protein was secreted irrespective of the treatment.
In addition, the results of our experiments examining the effect of
GM-CSF on the level of steady state mRNA for IL-8 and MIP-1 provide
further support for an effect predominantly at a post-transcriptional
or translational level, as pretreatment with GM-CSF failed to increase
the level of steady state mRNA for either chemokine induced by EBV.
Overall, these results suggest that GM-CSF enhances translation of both
IL-8 and MIP-1 protein in response to EBV but also increases the
proportion of IL-8 being secreted.
Induction of chemokine gene expression in neutrophils does not appear to require interaction of the cell with infectious EBV. Noninfectious particles such as the UV-irradiated B95-8 strain of EBV (otherwise infectious) were as potent as the untreated infectious EBV at inducing chemokine gene expression in neutrophils (data not shown). However, heat-inactivated EBV was significantly less potent than intact EBV, suggesting that the viral particles must be structurally intact for chemokine gene expression to occur (data not shown). In other studies, we have postulated that this may reflect a requirement for an interaction between gp350 of EBV and the neutrophil surface for stimulation to occur, a notion that is supported by the observation that the mAb 72A1, which binds to gp350, significantly reduces the ability of EBV to induce chemokine gene expression (20, 21). At this stage, however, the identity of the surface structure(s) with which EBV intereacts on the neutrophil surface is not known. It is clear that the known receptor for EBV, CD21 (CR2), is not expressed on neutrophils (20), leading to the suggestion of another receptor for EBV on neutrophils. In support of this possibility is the observation that another receptor for EBV appears to exist on other cells (39, 40).
Despite considerable research, the mechanism by which GM-CSF primes neutrophils for increased responsiveness to subsequent stimulation is unclear. Pretreatment with GM-CSF has been shown to increase a wide range of neutrophil functions such as calcium mobilization, the respiratory burst, 5-LO product synthesis, phagocytosis, tyrosine phosphorylation, and phospholipase D activation in response to a wide range of agonists including soluble chemotactic factors and particulate stimuli such as inflammatory microcrystals and zymosan (6, 33, 34, 35, 36, 41, 42). The present study is the first demonstration that GM-CSF primes neutrophils (or any cell type) for increased responsiveness to a virus. However, whether the signal transduction cascade used by EBV to activate chemokine gene expression in neutrophils is the same as that employed by these other agonists remains to be established. It is possible that pretreatment with GM-CSF increases the expression or signal transducing potential of the receptor(s) used by EBV to activate neutrophils, although as previously stated, such a receptor(s) remains to be identified. An increase in receptor expression, or in the binding of EBV to the neutrophil surface following treatment of the cells with GM-CSF, as is suggested by the results of the present study, could account, at least in part, for an increased response. However, it must be noted that whether GM-CSF primes neutrophils for increased responses to other agonists by increasing receptor expression is controversial (43, 44). In any case, other data have shown that GM-CSF enhances intracellular signaling at sites distal to receptor occupancy (45), and with respect to EBV signal transduction, such potential sites of action remain to be identified.
The overall biologic significance of the observation that GM-CSF
enhances the ability of EBV to induce chemotactic factor production by
neutrophils is unclear. In previous studies, we have shown that
interaction between EBV and neutrophils leads to expression of the IL-1
family of cytokines (IL-1 and -ß and IL-1Ra) in such a way as to
favor the production of IL-1Ra (21). We have postulated that such an
outcome may be immunosuppressive, which may be advantageous to the
virus. In the present study, we have shown that GM-CSF enhances
secretion of the chemokines IL-8 and MIP-1 and the 5-LO product
LTB4 by neutrophils in response to EBV. At the present
time, whether this observation is relevant to host defense against EBV
in particular or viruses in general is unknown. If it is relevant to
host defense, whether it is advantageous to the virus in terms of
evading host defense is also unclear. Together, these three products
are major chemotactic factors for phagocytes (11, 14, 46), and MIP-1
has been reported in vitro to be chemotactic for T cells and B cells
(12, 13). In terms of what is understood concerning the importance of
the inflammatory response in resolution of infection, it is likely that
production of chemotactic factors that increase recruitment of
phagocytes with APC potential would be disadvantageous to the virus. On
the other hand, B cells are the major cellular target of EBV, and
MIP-1, which has been shown in vitro to be chemotactic for B cells,
may enhance recruitment of B cells to the focus of infection. This
could increase EBV infectivity. Further studies will be required to
address this issue.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean Gosselin, Viral Immunology Laboratory, Centre de Recherche du CHUL, 2705 boulevard Laurier, Room T 1-49, Sainte-Foy (Québec), G1V 4G2, Canada.
3 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; MIP-1, macrophage inflammatory protein 1; LTB4, leukotriene B4; IL-1Ra, IL-1R antagonist; IM, infectious mononucleosis; RP-HPLC, reverse phase HPLC; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 5-LO, 5-lipoxygenase.
Received for publication May 27, 1997. Accepted for publication November 13, 1997.
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References |
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and MIP-1ß. Science 260:355.. J. Exp. Med. 178:63.This article has been cited by other articles:
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H. Shinohara, S. Yano, C. D. Bucana, and I. J. Fidler Induction of Chemokine Secretion and Enhancement of Contact-Dependent Macrophage Cytotoxicity by Engineered Expression of Granulocyte-Macrophage Colony-Stimulating Factor in Human Colon Cancer Cells J. Immunol., March 1, 2000; 164(5): 2728 - 2737. [Abstract] [Full Text] [PDF]
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