Download Herpesvirus Seropositivity in Childhood Monocyte-Induced Associates with Decreased Production

Herpesvirus Seropositivity in Childhood
Associates with Decreased Monocyte-Induced
NK Cell IFN- γ Production
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of August 30, 2013.
Shanie Saghafian-Hedengren, Yvonne Sundström, Ebba
Sohlberg, Caroline Nilsson, Annika Linde, Marita
Troye-Blomberg, Louise Berg and Eva Sverremark-Ekström
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 182:2511-2517; ;
doi: 10.4049/jimmunol.0801699
http://www.jimmunol.org/content/182/4/2511
The Journal of Immunology
Herpesvirus Seropositivity in Childhood Associates with
Decreased Monocyte-Induced NK Cell IFN-␥ Production1
Shanie Saghafian-Hedengren,2,3* Yvonne Sundström,3* Ebba Sohlberg,* Caroline Nilsson,†
Annika Linde,‡ Marita Troye-Blomberg,* Louise Berg,4§ and Eva Sverremark-Ekström4*
T
he viruses EBV and CMV belong to the herpesvirus
family and are widely spread in human populations.
Primary infections with EBV and CMV often occur
during early childhood and are usually asymptomatic or mild.
After primary infection, EBV and CMV establish latency and
persist for the lifetime of the host. Seropositivity for these viruses can vary among populations dependent on their socioeconomic background, and young children in industrialized countries seem more protected from early infection (1, 2). Latently
infected memory B cells are believed to constitute the main site
for EBV persistence (3), whereas the location for carriage of
CMV DNA is less clear but has been shown to include cells of
the myeloid lineage (4).
Because Ag-specific immune responses require time to develop
and expand, components of innate immunity play an important role
*Department of Immunology, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden; †Department of Clinical Science and Education, Sodersjukhuset,
Karolinska Institutet and Sachs’ Children⬘s Hospital, Stockholm, Sweden; ‡Department of Epidemiology, Swedish Institute for Infectious Disease Control, Solna, Sweden; and §Department of Microbiology, Tumour and Cell Biology, Strategic Research
Center Integrative Recognition in the Immune System, Karolinska Institutet, Stockholm, Sweden
Received for publication May 27, 2008. Accepted for publication December 13, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Swedish Research Council Grants 74X-15160-03-2,
57X-15160-05-2, and K2006-74X-15139-03-02); European Union Grants LSHP-CT
2004 and 503578; the Vardal Foundation; the Swedish Asthma and Allergy Association’s Research Foundation; the Swedish Medical Society; and the Golden Jubilee
Memorial, Crown-Princess Lovisa and Axel Tielman, Golje, Hesselman, Åhlén, and
Apotekare Hedberg foundations.
in the early phase of infections. NK cells are activated early after
a viral infection, and one of the central mechanisms by which they
mediate antiviral activity is through IFN-␥. This cytokine is a potent activator of APCs by influencing their maturation and antiviral
functions (5, 6). IFN-␥ can also act as an immunoregulatory cytokine in that it plays a central role in the shaping of Th1 immune
responses (7).
Although NK cells can exert immediate attacks, they generally
do not become fully functional unless triggered by cytokines that
are released upon recognition of viruses or viral components (6).
Among these cytokines are the proinflammatory ILs, IL-12, IL-15,
and IL-18, which are produced largely by accessory cells, such as
dendritic cells (DC)5 and monocytes (5, 6, 8, 9). In addition to
soluble factors, NK cells can become activated by a number of
contact-dependent mechanisms provided by accessory cells, and
numerous studies have investigated such interactions between NK
cells and DCs (10 –12).
NK cell and monocyte interactions have received less interest,
although it has been demonstrated that depletion of CD14⫹ cells
markedly reduces IFN-␥ production by NK cells (13, 14). In addition, blocking of the costimulatory molecules CD40 (15), CD80,
and CD86 (16) on activated monocytes has been shown to decrease NK cell IFN-␥ production. However, distinction of monocytes on the basis of CD14 expression alone might not be optimal
for examining their interactions with other cells, given that monocytes represent a heterogeneous population with functional differences. Monocytes are roughly divided into two major subsets and
are classified based on differential expression degrees of CD14 and
CD16 (Fc␥RIII). The larger subset is CD14⫹⫹CD16⫺ (classical
2
Address correspondence and reprint requests to Dr. Shanie Saghafian-Hedengren,
Stockholm University, Department of Immunology, Svante Arrheniusväg 16-18, 106
91 Stockholm, Sweden. E-mail address: [email protected]
3
Shared first authorship.
4
Shared last authorship.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801699
5
Abbreviations used in this paper: DC, dendritic cell; PGN, peptidoglycan; SN, seronegative; SP, seropositive; GeoMFI, geometrical mean fluorescence intensity.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
Downloaded from http://www.jimmunol.org/ at Stockholm University on August 30, 2013
EBV infection is inversely associated with IgE sensitization in children, and this association is further enhanced by CMV coinfection. In mice, herpesvirus latency causes systemic innate activation and protection from bacterial coinfection, implying the
importance of herpesviruses in skewing immune responses during latent infection. Early control of viral infections depends on
IFN-␥ release by NK cells, which generally requires the presence of accessory cells. We investigated IFN-␥ production by NK cells
in PBMCs from children seropositive (SP) for EBV alone, for both EBV and CMV, or seronegative for both viruses. The ability
of classical (CD14ⴙⴙCD16ⴚ) and proinflammatory (CD14ⴙCD16ⴙ) monocytes to induce autologous NK cell IFN-␥ was studied
by coculture experiments with enriched CD3ⴚCD56ⴙ cells. Transwell experiments were used to evaluate how monocytes interact
with NK cells to induce IFN-␥ synthesis. SP children had a significantly reduced proportion of IFN-␥ⴙ NK cells and cognate
intracellular IFN-␥ levels, which was more pronounced in CMV-coinfected subjects. Also, resting PBMCs of SP children displayed
lower proportions of proinflammatory monocytes. IFN-␥ production by NK cells was dependent on interactions with monocytes,
with the proinflammatory subset inducing the highest IFN-␥. Finally, SP children had markedly lower levels of plasma IFN-␥,
concurrent with in vitro findings. Herpesvirus infections could be one contributing factor for maturation toward balanced Th1Th2 responses. Our data indicate that early infection by herpesviruses may affect NK cell and monocyte interactions and thereby
also influence the development of allergies. The Journal of Immunology, 2009, 182: 2511–2517.
HERPESVIRUS IN CHILDHOOD ASSOCIATES WITH LOWER INNATE IFN-␥
2512
Table I. Subject demographics
CMV⫺EBV⫺ Childrena,b CMV⫹ and/or EBV⫹ Childrena EBV⫹CMV⫺ Childrenb EBV⫹CMV⫹ Childrenb
Group size (n)
Sex of child (n; girl/boy)
Delivery mode (n; vaginal/cesarean)
Maternal age at delivery (yr)c
Birth weight (kg)c
Gestation (wk)c
Exclusive breast-feeding (mo)c
Attending day care (n; yes/no)
Age at day care start (mo)c
Housing (n; apartment/house)
Pets at home (n; dog/cat)
Siblings (n; 0/ⱖ1)
Maternal smoking (n; yes/no)
a
b
25
9/16
22/3
28 (21–35)
3.7 (3.0 – 4.8)
40 (38 – 43)
4.5 (1–10)
23/2
18 (14 –21)
13/12
1/1
12/13
3/22
16
7/9
14/2
29 (21–35)
3.6 (3.0 – 4.8)
40 (39 – 43)
4 (1–10)
15/1
18 (14 –21)
7/9
1/1
7/9
2/14
9
2/7
8/1
28 (23–35)
3.9 (3.4 – 4.2)
40 (38 – 42)
4.5 (1–5)
8/1
17 (14 –20)
6/3
0/0
5/4
1/8
pb
NS
NS
NS
0.01
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.03
NS
NS
NS
NS
NS
NS
NS
NS
NS
Statistical comparison between two groups.
Statistical comparison between three groups.
Median (range).
monocytes), whereas the remaining subset is CD14⫹CD16⫹ (nonclassical or proinflammatory monocytes) (17, 18). Proinflammatory monocytes express higher levels of MHC class II and CD86
(17, 18) and are more likely to differentiate into DCs (19). They
also produce larger amounts of proinflammatory cytokines and
lower IL-10 compared with classical monocytes (20, 21). Alternatively, CD14⫹CD16⫹ monocytes are suggested to be the precursors of resident myeloid cells in noninflamed tissues (22), and
activated DCs derived from human CD16⫹ monocytes seem to
preferentially promote Th2 responses (23, 24).
Our group has previously reported an inverse association between EBV seropositivity and IgE sensitization in 2-year-old children, which seems to be augmented by the presence of CMV coinfection (25). When investigating plausible mechanisms behind this
protection in the T cell compartment, an overall increased cytokine
production in PHA-stimulated PBMCs in EBV-seropositive (SP)
2-year-old subjects was observed, whereas CMV infection was
associated with a bias toward a Th1-type cytokine profile (26).
This would suggest that both EBV and CMV could influence the
early life development of functionally diverse adaptive immune
responses.
Innate responses can greatly influence the generation and maintenance of subsequent adaptive effector functions (27, 28). A recent study in mice demonstrated that latency with herpesviruses is
associated with increased levels of circulating IFN-␥ and a stronger macrophage activity (29). However, corresponding data on humans are lacking. The objective of this study was therefore to
examine innate responses in relation to EBV and CMV seropositivity in 2-year-old children. We have studied monocyte-induced
NK cell IFN-␥ production in PBMCs of children with a positive or
negative serostatus for these herpesviruses and demonstrate that
seropositivity associates with decreased production of IFN-␥ by
NK cells, lower IFN-␥ levels in plasma, and a lower proportion of
CD14⫹CD16⫹ monocytes.
Materials and Methods
EBV and CMV (n ⫽ 26) and the other group (n ⫽ 25) with subjects SP for
either EBV alone (n ⫽ 16) or both EBV and CMV (n ⫽ 9). Further, buffy
coats from healthy adult blood donors were obtained from the blood bank
at Karolinska University Hospital (Stockholm, Sweden). This study was
approved by the Human Ethics Committee at Karolinska University Hospital (Huddinge, Sweden) and Karolinska Institutet, and the parents provided their informed consent.
Separation of plasma and mononuclear cells from blood, cell
isolation, and culture
Following collection of venous blood, plasma was separated after centrifugation and stored at ⫺70°C. PBMCs were isolated by Ficoll-Paque (Pharmacia-Upjohn) gradient centrifugation and cryopreserved in liquid nitrogen. For the in vitro experiments, PBMCs were thawed, then washed twice
in RPMI 1640-HEPES (Life Technologies), and resuspended in RPMI
1640 supplemented with 10% heat-inactivated FCS (HyClone Laboratories), L-glutamine (2 mM/L), penicillin G sodium (100 U/ml), and streptomycin sulfate (100 ␮g/ml; Merck) to a concentration of 106 cells/ml and
cultured for 24 h in 48-well (Corning) or 96-well (Sarstedt) flat-bottom
plates. PBMCs were cultured in medium alone or with IL-15 (20 ng/ml;
R&D Systems), peptidoglycan (PGN; 1 ␮g/ml; Staphylococcus aureus;
Fluka; Sigma-Aldrich) or IL-15 and PGN in combination. Supernatants
were stored at ⫺85°C until analysis. Where indicated, CD3⫺CD56⫹ NK
cells, CD14⫹ monocytes or subpopulations of monocytes (CD14⫹⫹
CD16⫺ and CD14⫹CD16⫹) were separated from PBMCs of healthy adult
donors, using MACS isolation kits according to the manufacturer’s instructions (Miltenyi Biotec). Enriched monocytes and autologous NK cells were
cultured alone or cocultured with or without transwell inserts (ratio, 1:1;
Corning) and treated with either medium or a combination of IL-15 and
PGN, as described above, during 24 or 44 h.
Flow cytometry
FITC-, PE-, PerCP-, or allophycocyanin-conjugated or biotinylated mAbs
were used for cell staining of CD3, CD56, CD16, CD14, CD11c, CD80,
CD86, and IFN-␥ according to standard procedures. The biotinylated primary Abs CD80 and CD86 were used in combination and detected with
streptavidin-PerCP conjugate. To detect intracellular IFN-␥, GolgiStop
was added during the last 4 h of incubation. After surface labeling, cells
were fixed, permeabilized, and stained for IFN-␥. Data were acquired and
analyzed with a FACSCalibur flow cytometer and CellQuestPro version
5.2.1 software, all from BD Biosciences. Intracellular IFN-␥ levels are
presented as geometrical mean fluorescence intensity (GeoMFI) values.
Characterization and serostatus of subjects
IL-6, IL-10, IL-12p70, IFN-␥, and TGF-␤ ELISA
A total of 51 IgE-nonsensitized 2-year-old children (Table I) were selected
from an earlier described family cohort (25). The presence of serum IgG
against EBV capsid Ag was determined according to previously published
immunofluorescence assays (30) where a specific fluorescence at a dilution
of 1/20 was regarded as a sign of seropositivity. IgG seropositivity to CMV
was determined by ELISA using purified nuclear Ags from CMV cultivated in human fetal lung fibroblasts (31), where samples with an OD ⬎0.2
at a dilution of 1/100 were considered positive. Subjects were divided into
two groups, one group with children that were seronegative (SN) for both
To determine IL-6, IL-10, IL-12p70, and IFN-␥ concentrations in cell supernatants or in blood plasma; Abs against IL-6, IL-10, IL-12p70, and
IFN-␥ (Mabtech); human rIL-6, rIL-10, rIL-12p70, and rIFN-␥ (National
Biological Standards Board); streptavidin-alkaline phosphatase-conjugated
Abs (Mabtech); and p-nitrophenyl phosphate (Sigma-Aldrich) were used.
TGF-␤ was measured with Quantikine ELISA kits (R&D Systems) according to the manufacturer’s instructions. The detection limit was 10
pg/ml for IL-6, IL-10, IL-12p70, and IFN-␥ and 4.6 pg/ml for TGF-␤
(mean value), respectively.
Downloaded from http://www.jimmunol.org/ at Stockholm University on August 30, 2013
c
26
16/10
23/3
32 (23– 42)
3.5 (2.7– 4.4)
40 (36 – 43)
4 (0 –10)
23/3
19 (14 –24)
14/12
2/5
16/10
1/25
pa
The Journal of Immunology
2513
The SP group of children had a significantly lower proportion of
IFN-␥⫹ NK cells among total NK cells ( p ⫽ 0.019; Fig. 1C)
accompanied by decreased levels of intracellular IFN-␥ measured
as GeoMFI ( p ⫽ 0.035; Fig. 1D) after stimulation with IL-15 and
PGN than did SN children. When SP subjects were further divided
into EBV⫹CMV⫺ and EBV⫹CMV⫹ subgroups, CMV coinfection
was associated with the lowest fraction of IFN-␥⫹ NK cells ( p ⫽
0.011, Fig. 1E) and the lowest levels of intracellular IFN-␥ ( p ⫽
0.010; Fig. 1F). This pattern was also observed for IFN-␥ detected
by ELISA in culture supernatants ( p ⫽ 0.039; Fig. 2), although no
significant differences were found when dividing the children only
FIGURE 1. IFN-␥ production capacity by PBMCs from 2-year-old children after 24 h of culture with indicated stimuli. A, Proportion of IFN-␥producing CD3⫺CD56⫹ NK cells among total NK cells; B, proportion of
IFN-␥-producing CD3⫹ T cells among total T cells; C and E, proportion
IFN-␥⫹ NK cells among total NK cells; D and F, GeoMFI of IFN-␥ in
gated NK cells after IL-15 plus PGN stimulation. Children were categorized in C and D as seronegative (SN, n ⫽ 26) or seropositive for EBV or
for EBV and CMV (SP, n ⫽ 25) and the seropositive group is subdivided
in E and F as EBV⫹CMV⫺ (n ⫽ 16) and EBV⫹CMV⫹ (n ⫽ 9). MFI,
mean fluorescence intensity.
Statistical analysis
The Mann-Whitney U test or Fisher’s exact test was used to evaluate
differences between sample groups. Cuzick’s nonparametric test was
used to evaluate trends. Outliers are not shown in figures. Data in the
text indicate median (numbers in parentheses, range). Statistical analyses were performed with Statistica 7.1 (Statsoft) and Stata 8 Software
(Stata) at the Division of Mathematical Statistics (Stockholm University, Stockholm, Sweden). p values below 0.05 were considered statistically significant.
Results
Herpesvirus seropositivity is associated with decreased IFN-␥
production by NK cells
The IFN-␥ production capacity was examined in 2-year-old subjects, either SP for EBV, EBV and CMV or SN for both viruses.
PBMCs were stimulated in vitro for 24 h with IL-15 or PGN or
with IL-15 and PGN in combination and stained for intracellular
IFN-␥. The flow cytometric analysis showed that only a combination of IL-15 and PGN induced effective IFN-␥ production. NK
cells represented the major IFN-␥⫹ cell population (Fig. 1A),
whereas only very low proportions of IFN-␥⫹CD3⫹ cells were
detected (Fig. 1B).
FIGURE 3. Monocyte subset distribution and phenotype after 24 h of
culture in medium alone (䡺) or with IL-15 plus PGN (u). A, Proportion
CD14⫹CD16⫹ monocytes of all CD14⫹ cells. B, GeoMFI of CD11c on
CD14⫹CD16⫹CD80/CD86⫹ monocytes. Children were categorized as seronegative (SN, n ⫽ 26) or seropositive for EBV or EBV and CMV (SP,
n ⫽ 25).
Downloaded from http://www.jimmunol.org/ at Stockholm University on August 30, 2013
FIGURE 2. IFN-␥ release by PBMCs from 2-year-old children after
24 h of culture with IL-15 plus PGN. Children were categorized as SN (n ⫽
24) or as EBV⫹CMV⫺ (n ⫽ 14) and EBV⫹CMV⫹ (n ⫽ 9).
2514
HERPESVIRUS IN CHILDHOOD ASSOCIATES WITH LOWER INNATE IFN-␥
according to their positive or negative serostatus ( p ⫽ 0.072; data
not shown).
SP and SN children did not differ in NK cell IFN-␥ production
after 24 h of culture in medium alone neither in the proportion of
IFN-␥⫹ NK cells (1% (0 –1) and 1% (0 –1), respectively) nor in
intracellular levels of IFN-␥, measured as GeoMFI, (8 (6 –18) and
8 (6 –31), respectively). IFN-␥⫹CD3⫹ cell proportions did not differ between SP and SN children (medium alone: 0% (0 –1) and 0%
(0 –1), respectively; IL-15 and PGN: 2% (0 –27) and 3% (0 – 67),
respectively), nor did the CD3⫹ IFN-␥ levels measured as
GeoMFI (medium alone: 8 (6 –12) and 8 (6 –11), respectively;
IL-15 and PGN: 9 (7–19) and 9 (7–21), respectively). There were
no significant differences between SP and SN children regarding
total NK cells of all lymphocytes (5% (2–21) and 5% (2–12),
respectively), CD56bright cells of all NK cells (4% (2–13) and 4%
(1–11), respectively), or total CD3⫹ cells of all lymphocytes (78%
(39 – 86) and 77% (50 – 88), respectively) after 24 h in medium.
Herpesvirus seropositivity associates with phenotypically
altered CD14⫹CD16⫹ monocytes
Next, we wanted to examine if herpesvirus seropositivity also affected monocyte characteristics. There was a clear tendency toward a decreased ratio of CD14⫹CD16⫹ monocytes after 24 h in
medium in SP compared with SN children ( p ⫽ 0.058; Fig. 3A),
although no differences were detected upon IL-15 and PGN stimulation (data not shown). When further assessing the phenotype of
the CD14⫹CD16⫹ subset, we found that CD11c expression on
CD14⫹CD16⫹CD80/CD86⫹ monocytes was significantly lower
in SP children after 24 h in medium alone or with stimulation ( p ⫽
0.042 and p ⫽ 0.003, respectively; Fig. 3B).
To assess monocyte function, a set of mainly monocyte-derived
cytokines was measured in cell culture supernatants. There were
FIGURE 5. Blood plasma levels of IFN-␥. Children were categorized in
A as SN (n ⫽ 26) or SP for EBV or EBV and CMV (n ⫽ 25) and the SP
group is further subdivided in B as EBV⫹CMV⫺ (n ⫽ 16) and
EBV⫹CMV⫹ (n ⫽ 9).
no significant differences in IL-6 release in culture supernatants
between SP and SN children (data not shown), although there was
a tendency for the EBV⫹CMV⫹ children to produce the lowest
amounts of IL-6 after stimulation, in analogy with the results for
IFN-␥. There was a positive correlation between the supernatant
IL-6 and IFN-␥ levels after challenge (rs ⫽ 0.66; p ⬍ 0.001).
There were no differences in IL-10 or TGF-␤ levels between the
groups (data not shown), and the levels of IL-12p70 in cell culture
supernatants were below the detection limit of our assay.
NK cell IFN-␥ production is more potently induced by
CD14⫹CD16⫹ monocytes
Given that the proportion of CD14⫹CD16⫹ monocytes was reduced in SP children, we wanted to address their potential contribution to the observed NK cell IFN-␥ responses. Consistent with
previous findings (13, 14), substantial IFN-␥ levels in the culture
supernatants were detected only in stimulated cocultures of
CD14⫹ cells and NK cells, whereas stimulation of either cell type
alone resulted in barely detectable levels of IFN-␥ (Fig. 4A). In
addition, IFN-␥ levels detected in monocyte and NK cell cocultures were comparable with those detected in PBMC cultures (Fig.
4A), indicating that the monocyte is the main NK cell-activating
cell type among PBMCs. We observed markedly higher levels of
IFN-␥ in cocultures of NK cells and CD14⫹CD16⫹ monocytes, as
compared with NK cell cocultures with CD14⫹⫹CD16⫺ cells
(Fig. 4B). In agreement with other studies (13–16, 32), when enriched monocytes and NK cells were cocultured but separated
by transwell inserts, the levels of IFN-␥ were decreased, indicating that cell-to-cell contact was required for optimal IFN-␥
Downloaded from http://www.jimmunol.org/ at Stockholm University on August 30, 2013
FIGURE 4. Influence of monocytes and monocyte subsets on NK cell
IFN-␥ production in culture with medium alone (䡺) or with IL-15⫹PGN
(f). A, IFN-␥ release from enriched CD3⫺CD56⫹ NK cells or total
CD14⫹ population alone, or in cocultures (NK:CD14⫹) or from PBMCs
after 24 h. B, IFN-␥ release from cocultures of enriched CD3⫺CD56⫹ NK
cells with classical (NK:CD14⫹⫹CD16⫺) or proinflammatory (NK:CD14⫹
CD16⫹) monocytes after 44 h. Median ⫾ SD of four to six (A) or three (B)
independent experiments from healthy adult donors. Vertical line through
bars, assay detection limit.
The Journal of Immunology
production by NK cells (inhibition range, 40 –55% from three
separate experiments).
Herpesvirus seropositivity in children is associated with
reduced levels of plasma IFN-␥
We further investigated whether the observed in vitro differences
in IFN-␥ production between the groups of children were evident
also in vivo and determined plasma IFN-␥ levels. In accordance
with our in vitro results, we found significantly lower levels of
circulating IFN-␥ in the plasma of the SP children ( p ⫽ 0.048, Fig.
5A). Again, the same pattern was found when the SP subjects were
subgrouped in which CMV coinfection was associated with the
lowest IFN-␥ levels ( p ⫽ 0.015; Fig. 5B). We also quantified IL-6
in plasma but found no significant differences between the groups.
Similarly to culture supernatants, there was a strong correlation
between plasma IFN-␥ and IL-6 (rs ⫽ 0.90; p ⬍ 0.001).
Throughout the process of virus-host coevolution, herpesviruses
have developed an array of immunomodulatory mechanisms to
avoid detection and destruction by the host’s immune system. Although loss of immune control can lead to herpesvirus reactivation
from latency and result in serious disease (2– 4), delayed primary
infection with herpesviruses in affluent societies (1, 2) coincides
with higher incidence of allergic disorders (32), which in contrast
indicates a beneficial role for these viruses.
In connection with this, our group has previously reported an
inverse association between EBV seropositivity and IgE sensitization in 2-year-old children (25). Recent observations from our laboratory did not provide support for a Th1-biased cytokine profile in
EBV SP children (26), which could have explained the protection
against the allergic phenotype (32, 33). However, latent herpesvirus infections in mice result in potentiated innate activity and increased systemic IFN-␥ levels (29). To elucidate the mechanism
behind protection against IgE sensitization in SP infants, we focused on monocyte and NK cell interplay given that their interaction has been suggested to play a role in the early control of viral
infections and might be important for the initiation and shaping of
subsequent adaptive responses (10, 12).
The cooperation between accessory cells and NK cells has recently been investigated in relation to diseases such as malaria (13,
14), rheumatoid arthritis (34), atopic dermatitis (35), and respiratory allergic disorders (36). To our knowledge, we are the first to
examine monocyte-induced IFN-␥ synthesis in NK cells in relation
to early life viral infections. We demonstrate that EBV seropositivity in infants is associated with reduced monocyte-induced NK
cell IFN-␥ production, with regard to both the proportion of
IFN-␥⫹ NK cells and to their cognate intracellular IFN-␥ levels.
This reduction was even more pronounced in children who were
SP for both EBV and CMV. Data on intracellular NK cell and cell
culture IFN-␥ paralleled, which strongly argues against different
kinetics in the synthesis of this cytokine between the groups. The
activation degree of CD14⫹ cells after PGN challenge has been
shown to correlate with released IL-6 (37). Interestingly, a strong
correlation was found between IL-6 and IFN-␥ levels in culture
supernatants after stimulation although no differences in IL-6 levels were seen between the groups.
PBMCs from SP children showed a strong tendency toward a
lower proportion of proinflammatory monocytes. When assessing
their functional properties, we found that they triggered IFN-␥
release by NK cells more potently than classical monocytes. This
effect could potentially be due to an increased capacity to secrete
IL-12 (38), which is a key inducer of IFN-␥ responses (39), but
levels of IL-12p70 in cell culture supernatants were below the
detection limit of our assay. The lower CD14⫹CD16⫹ ratio in SP
children could not be explained by decreased production of TGF-␤
or IL-10, cytokines that have been shown to induce CD16 expression on monocytes (40, 41). However, we cannot rule out that the
EBV and CMV encoded homologs of human IL-10 could have
affected the quantification of IL-10 in plasma (42). To our knowledge, IFN-␥ has no clear involvement in regulation of CD16 on
monocytes (43, 44). Furthermore, NK cells can be regulatory by
killing immature DCs (45) or overactivated macrophages (46), but
whether preferential elimination of the CD14⫹CD16⫹ cells could
be an explanation behind decreased proportions in SP children
remains to be elucidated.
Interestingly, plasma IFN-␥ levels in SP children were significantly lower than in their SN counterparts, concurrent with our in
vitro data. Again, CMV coinfection was associated with the lowest
levels of IFN-␥. The reason behind this finding is unknown but is
indicative of in vivo interactions between EBV and CMV with
immunomodulatory consequences, something which is supported
by a study from Aalto et al. (47) showing that primary CMV infection induced reactivation of latent EBV. Thus, in the doublepositive children, CMV coinfection may intensify EBV-induced
immune scarring. Furthermore, IL-6 and IFN-␥ levels correlated,
suggesting a possible in vivo monocyte and NK cell interaction.
The fact that the NK cell IFN-␥ production in vitro and plasma
levels of IFN-␥ paralleled could imply that NK cell-derived IFN-␥
was the major source of IFN-␥ in plasma. Indeed, T cells are also
important in controlling viral infections (48), but here their contribution to the plasma IFN-␥ is unclear. However, because the SP
subjects had lower IFN-␥ in their plasma, and we have observed an
overall increased cytokine production by PHA-stimulated PBMCs
from SP children (26), this strongly argues against T cells as the
major contributors to the differences in plasma IFN-␥ between the
groups.
A modulated accessory cell function due to herpesvirus infections may influence NK cell IFN-␥ responses in SP subjects. For
instance, infection of monocyte-derived DCs with CMV (49) and
HSV 1 (50) inhibits their maturation process as well as their release of inflammatory cytokines. Ex vivo data obtained here from
SP infants strengthens these findings and suggests that reduced
proportions of CD14⫹CD16⫹ cells might play a part in attenuated
NK cell IFN-␥ responses after pathogen encounter. In a model of
transendothelial trafficking, CD14⫹CD16⫹ cells showed a higher
likelihood to differentiate into DCs (19); suggesting that they could
represent physiologically relevant blood precursors of migrating
DCs, with the potential to interact with NK cells at sites of inflammation or in secondary lymphoid tissues (51). In addition to
the decreased ratio, CD14⫹CD16⫹ monocytes from SP subjects
who displayed an activated phenotype expressed lower levels of
CD11c, implying that a modulation of migratory behavior might
occur (52).
Exposure to pathogenic and nonpathogenic agents in childhood
strongly influences the progressive shift from the allergy-related
Th2 profile toward a balanced Th1-Th2 immunity (33). At first
sight, the present data seem difficult to reconcile with our previous
data of a high cytokine production in SP children (26). Intriguingly, the two studies indicate that EBV and CMV seropositivity is
associated with a lower NK cell but higher T cell cytokine response. A viral-induced modified balance between monocytic subsets might be one possible explanation behind this observation.
The finding of lower plasma IFN-␥ in SP children was unexpected since recent findings in mice with herpesvirus latency suggested the opposite (29). Besides, acute EBV infection in adults
causes mononucleosis, which is accompanied by higher in vivo
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Discussion
2515
2516
HERPESVIRUS IN CHILDHOOD ASSOCIATES WITH LOWER INNATE IFN-␥
Acknowledgments
A special thanks to Monica Nordlund, Anna Stina Ander, and Ann Sjölund
for their valuable assistance and to Jan-Olov Persson for performing statistical analyses.
Disclosures
The authors have no financial conflict of interest.
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