We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1α (MIP-1α). 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-1α 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-1α 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.

Granulocyte-macrophage CSF (GM-CSF)3 is a pleiotropic cytokine that induces the differentiation and proliferation of granulocyte and macrophage precursor cells. Over the last decade, many biologic activities of GM-CSF on neutrophils have been reported (reviewed in 1 . These include direct effects such as neutrophil degranulation, changes in receptor expression, and cytoskeletal rearrangement and shape change, as well as priming effects such as enhancing the ability of neutrophils to respond to a secondary stimulus. Among these, respiratory burst activity, intracellular Ca+2 mobilization, cytokine secretion, and leukotriene B4 (LTB4) synthesis are increased following stimulation by FMLP or platelet-activating factor (2, 3, 4, 5, 6, 7). These observations demonstrate that GM-CSF enhances the immune potential of neutrophils in host defense.

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-1α (MIP-1α), 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-1α, 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-α 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-1α and -β 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-1α, IL-2, IL-6 and IFN-γ 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-1β, IL-6, and TNF-α expression was strongly enhanced in EBV-infected cells (24, 25). Finally, in the same tissue samples, high levels of IL-1α and -β 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.

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.

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 × 107 transforming units (TFU)/ml. No IL-1α, IL-1β, TNF-α, GM-CSF, IL-8, or MIP-1α, as assessed by specific ELISA, was detected in the EBV preparations used.

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 × 104 monocytes/107 cells were used. Cell viability exceeded 97% as detected by trypan blue exclusion and lactate dehydrogenase activity.

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-1α levels were assessed using an ELISA kit purchased from R&D Systems (Minneapolis, MN). This MIP-1α assay had a minimal detectable level of 2 pg/ml and did not cross-react significantly with other known cytokines.

For these experiments, neutrophils were resuspended at 5 × 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 × 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).

Intracellular free calcium was monitored using the fluorescent probe fura-2/AM as previously described (30). Briefly, neutrophil suspensions (1 × 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 × 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).

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-1α 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.

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 × 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 analyses were performed using Student’s paired (two-tailed) t test, and significance was considered attained at p < 0.05.

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).

Table I.

Effect of GM-CSF on EBV-induced IL-8 production by neutrophilsa

Cell-Associated IL-8 (pg/ml)Secreted IL-8 (pg/ml)Total IL-8 (pg/ml)
Unstimulated 2,164 ± 430 89 ± 17 2,253 ± 443 
GM-CSF 10,716 ± 2,599* 913 ± 135** 11,629 ± 2,671* 
EBV 13,483 ± 3,927* 3,442 ± 1,017** 16,925 ± 4,938* 
GM-CSF + EBV 34,983 ± 6,351* 23,833 ± 9,356* 58,816 ± 15,230* 
Cell-Associated IL-8 (pg/ml)Secreted IL-8 (pg/ml)Total IL-8 (pg/ml)
Unstimulated 2,164 ± 430 89 ± 17 2,253 ± 443 
GM-CSF 10,716 ± 2,599* 913 ± 135** 11,629 ± 2,671* 
EBV 13,483 ± 3,927* 3,442 ± 1,017** 16,925 ± 4,938* 
GM-CSF + EBV 34,983 ± 6,351* 23,833 ± 9,356* 58,816 ± 15,230* 
a

Neutrophils were preincubated with or without 1 nM GM-CSF for 1 h at 37°C and further incubated with or without EBV. Cell-associated and secreted IL-8 were assessed by a specific ELISA. Results are expressed as pg/ml and are the mean ± SEM of six separate experiments. Values obtained for neutrophils incubated with GM-CSF or EBV alone were compared with those for unstimulated cells, and values for GM-CSF-treated neutrophils stimulated with EBV were compared with the sum of the values obtained when neutrophils were incubated with GM-CSF or EBV alone. *p < 0.05, **p < 0.01.

We next evaluated the effect of GM-CSF on EBV-induced MIP-1α production by neutrophils. Neutrophils incubated in medium alone failed to produce MIP-1α (Table II). Neutrophils were found to synthesize significant amounts of MIP-1α when incubated with EBV alone during 8 h, whereas GM-CSF alone exerted only a minor effect on MIP-1α production. In contrast to the effect observed with IL-8, approximately 96% of the MIP-1α produced in response to EBV was secreted. Pretreatment of neutrophils with GM-CSF before stimulation with EBV synergistically increased the production of MIP-1α compared with that induced by EBV and GM-CSF alone. In this case, the level of secreted MIP-1α was increased 2.2-fold.

Table II.

Effect of GM-CSF on the production of MIP-1α by neutrophils in response to EBVa

Cell-Associated MIP-1α (pg/ml)Secreted MIP-1α (pg/ml)Total MIP-1α (pg/ml)
Unstimulated 2 ± 1 3 ± 1 5 ± 1 
GM-CSF 3 ± 2 42 ± 14 45 ± 14 
EBV 40 ± 8* 867 ± 65** 907 ± 73** 
GM-CSF + EBV 31 ± 4 1944 ± 273* 1975 ± 274* 
Cell-Associated MIP-1α (pg/ml)Secreted MIP-1α (pg/ml)Total MIP-1α (pg/ml)
Unstimulated 2 ± 1 3 ± 1 5 ± 1 
GM-CSF 3 ± 2 42 ± 14 45 ± 14 
EBV 40 ± 8* 867 ± 65** 907 ± 73** 
GM-CSF + EBV 31 ± 4 1944 ± 273* 1975 ± 274* 
a

Neutrophils (107/ml) were preincubated with or without 1 nM GM-CSF for 1 h at 37°C and further incubated with or without EBV. Cell-associated and secreted MIP-1α were assessed by a specific ELISA. Results are expressed as pg/ml and are the mean ± SEM of four separate experiments. Values obtained for neutrophils incubated with GM-CSF or EBV alone were compared with those for unstimulated cells, and values for GM-CSF-treated neutrophils stimulated with EBV were compared with the sum of the values obtained when neutrophils were incubated with GM-CSF or EBV alone. *p <0.05, **p <0.01.

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.

FIGURE 1.

Biologic activity of the IL-8 secreted by GM-CSF-treated neutrophils in response to EBV. Supernatants from diluent 1- or GM-CSF-treated neutrophils incubated with EBV were collected and preincubated in the presence or absence of a monoclonal anti-human IL-8 Ab before testing their ability to induce the mobilization of intracellular free calcium in neutrophils. Intracellular free calcium was monitored as described in Materials and Methods. The arrows indicate the time of addition of supernatant from diluent-treated neutrophils (1) and GM-CSF-treated neutrophils incubated with EBV (2). The data presented are from a single experiment representative of three separate determinations.

FIGURE 1.

Biologic activity of the IL-8 secreted by GM-CSF-treated neutrophils in response to EBV. Supernatants from diluent 1- or GM-CSF-treated neutrophils incubated with EBV were collected and preincubated in the presence or absence of a monoclonal anti-human IL-8 Ab before testing their ability to induce the mobilization of intracellular free calcium in neutrophils. Intracellular free calcium was monitored as described in Materials and Methods. The arrows indicate the time of addition of supernatant from diluent-treated neutrophils (1) and GM-CSF-treated neutrophils incubated with EBV (2). The data presented are from a single experiment representative of three separate determinations.

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In previous studies, we have observed that EBV primes neutrophils for O

\(_{2}^{-}\)
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.

FIGURE 2.

GM-CSF enhances the priming effect of EBV on the production of LTB4 by neutrophils. Neutrophils were preincubated with or without 1 nM GM-CSF for 30 min at 37°C, further incubated with or without EBV for 30 min, and stimulated with or without a suboptimal concentration of A23187 (50 nM) for 5 min. Analyses of LTB4 were performed by RP-HPLC as described in Materials and Methods. Results are from one experiment (mean ± SD of triplicate incubations) and are representative of three separate experiments.

FIGURE 2.

GM-CSF enhances the priming effect of EBV on the production of LTB4 by neutrophils. Neutrophils were preincubated with or without 1 nM GM-CSF for 30 min at 37°C, further incubated with or without EBV for 30 min, and stimulated with or without a suboptimal concentration of A23187 (50 nM) for 5 min. Analyses of LTB4 were performed by RP-HPLC as described in Materials and Methods. Results are from one experiment (mean ± SD of triplicate incubations) and are representative of three separate experiments.

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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-1α synthesis in neutrophils treated with EBV or the control. These results confirm that the enhancing effects observed are dependent on GM-CSF.

Table III.

Specificity of the effect of GM-CSF on EBV-induced IL-8 and MIP-1α production by human neutrophilsa

Total IL-8 (pg/ml)Total MIP-1α (pg/ml)
Unstimulated (1) 3,132 ND 
 (2) 1,528 ND 
EBV (1) 20,300 744 
 (2) 22,010 808 
GM-CSF (1) 23,750 56 
 (2) 11,700 24 
GM-CSF + EBV (1) 81,500 2,012 
 (2) 55,000 2,812 
Anti-GM-CSF Abs (1) 3,200 ND 
 (2) 1,390 ND 
Neutralized GM-CSF (1) 3,380 ND 
 (2) 2,090 ND 
Neutralized GM-CSF+ EBV (1) 28,100 726 
 (2) 25,240 786 
Total IL-8 (pg/ml)Total MIP-1α (pg/ml)
Unstimulated (1) 3,132 ND 
 (2) 1,528 ND 
EBV (1) 20,300 744 
 (2) 22,010 808 
GM-CSF (1) 23,750 56 
 (2) 11,700 24 
GM-CSF + EBV (1) 81,500 2,012 
 (2) 55,000 2,812 
Anti-GM-CSF Abs (1) 3,200 ND 
 (2) 1,390 ND 
Neutralized GM-CSF (1) 3,380 ND 
 (2) 2,090 ND 
Neutralized GM-CSF+ EBV (1) 28,100 726 
 (2) 25,240 786 
a

Neutrophils were preincubated with or without 1 nM of active or mAb-neutralized GM-CSF for 1 h at 37°C and further incubated with or without EBV for 8 h. Total (cell-associated + secreted) IL-8 and MIP-1α were assayed by specific ELISA. Neutralized GM-CSF was obtained by incubating 2 volumes of neutralizing Abs with 1 volume of stock GM-CSF solution for 30 min at 37°C. ND, not detectable.

The steady state levels of IL-8 and MIP-1α 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-1α 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-1α, and GAPDH cDNA, respectively. Neutrophils incubated in medium alone failed to express detectable levels of IL-8 or MIP-1α. An increase in the level of IL-8 and MIP-1α 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-1α. Furthermore, treatment of neutrophils with GM-CSF before incubation with EBV did not modify the levels of IL-8 or MIP-1α mRNA compared with those obtained in response to either GM-CSF or EBV alone.

FIGURE 3.

Effect of GM-CSF on the accumulation of IL-8 and MIP-1α mRNA induced by EBV in neutrophils. Normal human blood neutrophils (107/ml) were preincubated with diluent or 1 nM GM-CSF for 1 h at 37°C and incubated in the presence or absence of EBV for 3 h at 37°C. Identical cell pellets equivalent to 40 × 106 neutrophils in each experimental condition were pooled and total RNA extracts prepared as detailed in Materials and Methods. Equivalent amounts of RNA (10 μg/lane) were size fractionated on denaturating agarose gel electrophoresis, blotted onto Hybond-N membranes, and sequentially probed with 32P-labeled IL-8, MIP-1α, and GAPDH cDNA. The results presented in this figure are from one experiment and are representative of three separate experiments.

FIGURE 3.

Effect of GM-CSF on the accumulation of IL-8 and MIP-1α mRNA induced by EBV in neutrophils. Normal human blood neutrophils (107/ml) were preincubated with diluent or 1 nM GM-CSF for 1 h at 37°C and incubated in the presence or absence of EBV for 3 h at 37°C. Identical cell pellets equivalent to 40 × 106 neutrophils in each experimental condition were pooled and total RNA extracts prepared as detailed in Materials and Methods. Equivalent amounts of RNA (10 μg/lane) were size fractionated on denaturating agarose gel electrophoresis, blotted onto Hybond-N membranes, and sequentially probed with 32P-labeled IL-8, MIP-1α, and GAPDH cDNA. The results presented in this figure are from one experiment and are representative of three separate experiments.

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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. 4,A). This increased to ∼71 ± 5% (n = 4) following a 1-h preincubation with GM-CSF (Fig. 4,B). 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. 4 C).

FIGURE 4.

Effect of GM-CSF on the binding of EBV to neutrophils. Neutrophils were pretreated with GM-CSF (1 nm) or diluent for 1 h at 37°C and processed for assessment of EBV binding by flow cytometry. A, Diluent-treated neutrophils; B, GM-CSF-treated neutrophils; C, neutrophils preincubated with unlabeled EBV before addition of FITC-EBV. The number in the lower righthand corner of each figure represents the percentage of positive cells. The dark peaks represent background binding of FITC diluent to neutrophils. Each histogram is representative of three other experiments performed with similar results. Fluorescence is expressed on a logarithmic scale.

FIGURE 4.

Effect of GM-CSF on the binding of EBV to neutrophils. Neutrophils were pretreated with GM-CSF (1 nm) or diluent for 1 h at 37°C and processed for assessment of EBV binding by flow cytometry. A, Diluent-treated neutrophils; B, GM-CSF-treated neutrophils; C, neutrophils preincubated with unlabeled EBV before addition of FITC-EBV. The number in the lower righthand corner of each figure represents the percentage of positive cells. The dark peaks represent background binding of FITC diluent to neutrophils. Each histogram is representative of three other experiments performed with similar results. Fluorescence is expressed on a logarithmic scale.

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Among other elements, regulation of the host response to infection involves generation of cytokines and chemotactic factors. Release of chemokines and the 5-LO product LTB4 by infected cells, or by cells such as neutrophils accumulating as part of the inflammatory response to infection, is likely to be a critical factor in the successful resolution of the infection. At such sites, neutrophils are also likely to be exposed to GM-CSF produced by macrophages, endothelial cells, activated T cells, or connective tissue cells such as fibroblasts. This cytokine has been shown to directly activate as well as to prime neutrophils for increased responsiveness to secondary signals (6, 33, 34, 35, 36). In the present study, we have investigated a possible interaction between GM-CSF and EBV in regulating chemotactic factor production by human neutrophils. Our results show that pretreatment of neutrophils with GM-CSF enhances the production of the chemokines IL-8 and MIP-1α 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-1α. As previously reported (38), GM-CSF induced significant expression of IL-8 but exerted a minor effect on MIP-1α 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-1α 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-1α, 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-1α 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-1α 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-1α and -β 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-1α 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-1α 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-1α, 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.

We thank Mrs. Pierrette Côté for excellent secretarial assistance.

1

This work was supported by a grant from the Medical Research Council of Canada (MRC) to J.G. and from the National Health and Medical Research Council of Australia to S.R.M. J.G. is a Scholar of the MRC. C.J.R. is the recipient of a Postdoctoral Fellowship from the MRC. B.L. is the recipient of a Postgraduate Studentship from the “Fonds pour la Formation de Chercheurs et à l’Aide à la Recherche” (FCAR).

3

Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; MIP-1α, macrophage inflammatory protein 1α; 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.

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