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Laboratory of Immunovirology, Department of Microbiology and Immunology, and Pediatric Research Center, University of Montreal and Sainte-Justine Hospital, Montreal, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix bundle cytokine family and possesses biological activities
similar to those of IL-2. Its ability to induce effectors of NK
activity suggests its involvement in innate immunity. In this study, we
analyzed the effect of different viruses (HSV, EBV, respiratory
syncitial virus, vesicular stomatitis virus, influenza virus, reovirus,
and Sendai virus) on the up-regulation of NK activity in vitro.
Exposure of human PBMC to the these viruses resulted in an immediate
up-regulation of NK activity of PBMC via IL-15 induction; this effect
was abrogated in the presence of mAbs to IL-15. Results of experiments
conducted in parallel using mAbs to IL-15, as well as to other
cytokines (IL-2, IL-12, IFN-
, and TNF-), clearly indicated that
IL-15 was specifically responsible for the observed effect.
Furthermore, supernatants of virus-infected PBMC cultures significantly
enhanced NK activity of uninfected PBMC in vitro. An increase of IL-15
protein levels 20 h postinfection was also confirmed in a bioassay
using the IL-2-dependent cell line CTLL. Kinetic analysis of IL-15 mRNA
expression using a semiquantitative RT-PCR revealed that the level of
IL-15 messages peaked at different time points (up to 12 h)
postinfection, depending on the nature of the virus. Taken together,
these results suggest that the IL-15 response of the host to viral
infection and the subsequent NK cell activation represent an important
effector mechanism of the innate immune surveillance of the host
against viral infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains of the IL-2 receptor for binding and signal transduction
(3). Both IL-15 and IL-2 share the ability to support the
growth of various T cell lines (1), Ag-dependent T cell
clones (Th0, Th1, and Th2) and activated normal T cells
(4). They both have a costimulatory activity for the
proliferation and Ig production of human tonsilar B cells, but IL-15
has no effect on resting B cells as tested in short-term incubation in
vitro (5). IL-15 is also an efficient activator of NK
cytotoxic activity (6). IL-15 was found to activate human
NK cells using components of the IL-2R and to synergize with IL-12 to
significantly potentiate NK cell production of IFN-
(6). Although IL-15 shares biological activities with
IL-2, there are several properties of IL-15 distinct from those of
IL-2. IL-15 uses a specific -chain of the IL-15R identified on a
murine Th2 cell clone, which is distinct from the -chain of the
IL-2R (7). In addition, although IL-2 is selectively
expressed in activated T cells, IL-15 mRNA has been found to be
constitutively expressed in several human tissues, including epithelial
cells, placenta, skeletal muscle, kidney, lung, heart, and activated
monocytes/macrophages (1). NK cells are important effectors in the natural immune response and play a major role as the first line of defense against viral, bacterial, and parasitic infections (8, 9). Through their non-MHC-restricted cytotoxicity, they can also kill tumor cells, thus providing the host with a defense mechanism that would target intracellular pathogens as well as cancerous cells (10). Furthermore, NK cells act via their FcRIII receptor (CD16) as effectors in the Ab-dependent cell-mediated cytotoxicity (ADCC)5 (11). This allows the immune system to build an adaptive immune response aimed at eradicating the pathogen (12, 13). Therefore, any defect in NK function would leave the host vulnerable to several major viral infections such as by herpesviruses (14) and HIV (15), hence contributing to viral pathogenesis (14, 16).
Recent studies in this laboratory have shown that infection of human PBMC with human herpesvirus (HHV)-6 and HHV-7 resulted in an immediate up-regulation of NK activity of these PBMC via IL-15 induction (17, 18). Earlier preliminary experiments using EBV, HSV, and HHV-6 have shown that this IL-15 induction and NK activation do not occur if the viral preparations are preincubated with human serum containing virus-neutralizing Abs and that virus Ab seropositive or seronegative status of the donor of PBMC had no effect on NK and IL-15 responses of PBMC to these viruses (L. Flamand and J. Menezes, unpublished results). Cytokines play an important regulatory role in the initiation and maintenance of the immune response. To gain a better understanding of the IL-15 response of the host to viral infection as part of an innate defense mechanism, we sought to determine whether different, unrelated viruses have the ability to enhance NK activity of human PBMC via IL-15 induction. Thus, we undertook a comparative study in which we investigated the following viruses belonging to different families, namely, influenza virus, HSV-1, EBV, reovirus, vesicular stomatitis virus (VSV), Sendai virus, and the respiratory syncitial virus (RSV), for their ability to up-regulate the NK cytotoxic activity of virus-infected PBMC in vitro. Here, we present data that suggest that up-regulation of NK activity via IL-15 induction represents an early effector mechanism of the host’s innate immune response to viruses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heparinized venous blood, freshly obtained from healthy donors, was centrifuged over a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient, and PBMC were collected as described (17). The separated PBMC were washed and cultured in RPMI 1640 culture medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FBS, 1.0% glutamine, 100 U/ml penicillin, 20 µg/ml streptomycin, and 1 µg/ml gentamicin.
Cell treatment
The PBMC (1 x 106 cells) were infected with optimal doses of viruses (as determined in preliminary experiments for maximal induction of NK cytotoxicity) or treated with mock-infected culture supernatant for 2 h at 37°C, washed with Hank’s buffer (Life Technologies), and then resuspended in 1 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS for 20 h at 37°C with or without a mAb to IL-15. After 20 h, cell-free supernatants from mock- and virus-treated PBMC were collected and kept at -80°C until used, and cells were prepared for the NK cytotoxicity assay as described (17).
Cell-free supernatants were added to untreated resting PBMC (24 h
postseparation) at 25% of the final volume, in the presence or absence
of a mAb to IL-15, as well as mAbs to other cytokines such as IL-2,
IL-12, IFN-, and TNF-, before mixing with the NK cell targets,
K562 cells. The NK activity of PBMC was tested in the presence of
recombinant human IL-15 (50 ng/ml) and anti-IL-15 Abs (10 µg/ml).
The choice of 50 ng/ml concentration of rIL-15 used in these
experiments was based on the fact that, in our preliminary experiments,
increasing concentrations of human rIL-15 from 10 to 50 ng/ml induced a
linear increase of cytotoxic activity, whereas no significant
differences were seen at concentrations between 50 and 100 ng/ml (data
not shown). This is also in agreement with our previously published
results (17).
Cell lines and viruses
The K562 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA). The CTLL-2 cell line was obtained from Dr. R.-P. Sékaly’s laboratory (Clinical Research Institute of Montreal and Department of Microbiology, University of Montreal). K562 cells were cultured in RPMI 1640 supplemented with 10% FBS, and CTLL-2 cells were cultured in RPMI 1640 with 5% FBS supplemented with 5 x 10-5 M 2-ME. Treatment of PBMC (1 x 106 cells) with each of the viruses used was as follows: 200 µl of EBV (B95-8 strain) preparation with a titer of 2 x 105 EBV nuclear Ag-inducing U/ml (19); 80 µl of HSV-1 at a multiplicity of infection of 50 PFU/cell (McIntyre strain) (19); 200 µl of human RSV, Long strain; ATCC) at a titer of 107 syncytia-forming units/ml (20); 200 µl of Sendai virus containing 750 hemaglutinating units in PBS (21); 200 µl of reovirus preparation (108 PFU/ml) (kindly provided by Dr. Guy Lemay, Department of Microbiology, University of Montreal); 50 µl of influenza virus A (H1N1) strain A1/FM/1/47 with a titer of 106.25 at a chick embryo infectious dose of 50/0.2 ml (ATCC); 200 µl of VSV 10-1 dilution 105 median tissue culture infectious dose (TCID50) kindly supplied by Dr. Youssef Elazhary (Faculty of Veterinary of Medicine, University of Montreal). Viral preparations were first titrated to determine the dose that showed the optimal NK-inducing activity (see below). Viral preparations were also tested for the presence of endotoxin by the Limulus amebocyte assay (Sigma Chemical Co, St. Louis, MO), and were found to contain <20 pg/ml of contaminating endotoxin.
NK cell cytotoxicity assay
All cytotoxicity assays were performed using either untreated resting PBMC or virus-infected PBMC (20 h postinfection: PI) as effectors. All assays were done using a standard 51Cr-release assay as described previously (17). Briefly, K562 target cells (1 x 106 cells/ml) were labeled by incubation with 100 µCi of 51Cr (DuPont/NEN, Boston, MA) for 1 h at 37°C. The radiolabeled target cells were then washed four times with RPMI 1640, and resuspended at a concentration of 1 x 105 cells/ml in RPMI 1640 with 10% FBS. K562 cells were then added into V-bottom wells and mixed with 0.05 ml PBMC (4 x 106 cells/ml) from healthy donors at an E:T ratio of 20:1 and incubated for 16 h at 37°C. Radioactivity was then measured using a gamma-counter (Wallac model 1272; LKB, Turcu, Finland). The percentage of cytotoxicity was calculated based on the following formula: [(cpm experimental - cpm spontaneous)/(cpm maximum - cpm spontaneous)] x 100. All these experiments were done in triplicate, and the results are presented as the mean ± SE of three independent determinations. The spontaneous cpm were determined by counting the radioactivity of the supernatant of the target cell suspensions, whereas the maximum cpm were determined from the radioactivity of the resuspended target cells with Triton-X (100 µl).
Abs and cytokines
Neutralizing mAbs to human IL-2, IL-12, and TNF- were
purchased from R&D Systems (Minneapolis, MN). Neutralizing mAbs to
IFN- were purchased from Genzyme (Boston, MA). Human rIL-15 and mAbs
to IL-15 were a gift from Immunex (Seattle, WA). All mAbs were used at
a concentration of 10 µg/ml, and IL-15 was used at a concentration of
50 ng/ml. The choice of this concentration (i.e., 10 µg/ml) of mAbs
in our experiments was established following preliminary experiments
(using HSV-1, HHV-6, and influenza virus) in which undiluted and
diluted preparations of mAbs were tested for their ability to inhibit
NK enhancement induced by different concentrations of supernatants from
virus-infected primary PBMC cultures; only mAbs to IL-15 were found to
significantly inhibit this enhancement, whereas Abs to other
above-listed cytokines and to IFN- and -ß had no such effect (Ref.
17 and J. Menezes, unpublished data). For anti-IL-15
mAbs, concentrations of 5 to 10 µg/ml produced maximum inhibitory
effect in all preliminary tests, including tests against 50 ng/ml of
human rIL-15 (data not shown). An additional consideration for the use
of the same concentration of mAbs in these experiments was also the
fact that some of these mAbs represent the needed control (negative)
Abs in relation to the inhibition that is observed upon neutralization
of IL-15 by anti-IL-15 mAbs.
CTLL assay
Supernatants from infected PBMC cultures (2 x 106 cells), obtained after 20 h incubation with each of the different viruses, were collected and tested for the presence of bioactive IL-15 by its ability to sustain the proliferation of the IL-2- and IL-15-responsive CTLL cell line (1). Treatment of supernatants with a mAb to IL-2 ascertained that proliferation of CTLL-2 cells was not due to IL-2 (data not shown). Cells were washed three times and incubated in RPMI 1640 medium containing 5 x 10-5 M 2-ME without FBS for 3 h at 37°C, in 5% CO2. CTLL cells (5 x 103 cells/100 µl) were then dispensed into 96-well round-bottom plates containing 100 µl of supernatant. After 66 h of incubation at 37°C in 5% CO2, cells were pulsed with 1 µCi of 3H/well (DuPont/NEN) for 6 h. Cells were then harvested on glass-fiber filter paper by an automated sample cell harvester (Tomtec, Orange, NJ) and dried. The incorporation of radioactivity was determined in a liquid scintillation counter. Results represent the mean of two replicate wells of two independent experiments and are expressed as cpm of [3H]thymidine incorporation.
Preparation of IL-15 mRNA and RT-PCR
PBMC (1 x 106 cells) were treated with different viruses for 2 h at 37°C, washed, and resuspended in 1 ml RPMI 1640 supplemented with 10% FBS. At various time intervals (2, 4, 8, 12, and 20 h) posttreatment, cells were counted and checked for mortality by trypan blue exclusion staining (<5%), and 5 x 105 cells were lysed and stored at -70°C until assayed for IL-15 mRNA expression.
Total mRNA was extracted from cells using a modified guanidium isothiocyanate procedure as described previously (22). All reagents used in this test were purchased from Life Technologies unless indicated otherwise. Total RNA extracted was finally resuspended in a total volume of 40 µl diethylpyrocarbonate-treated ddH2O. The samples were then treated with 10 U DNase I for 30 min at 37°C. The isolated RNA (from infected and mock-treated PBMC) was subjected to RT-PCR to determine the level of expression of IL-15 mRNA.
For first-strand cDNA synthesis, 4 µl of total RNA was reverse transcribed in a total volume of 10 µl containing 100 U recombinant Moloney murine leukemia virus reverse transcriptase, 2 µl 5x first-strand buffer (250 mM Tris-HCl, 375 mM KCl, and 15 mM MgCl2, pH 8.32), 1 µl of 1 mg/ml random hexamer primer (Pd N6), 0.01 M DTT, 0.5 µl of a 5 mM mixture of all four dNTP, and 30 U RNase inhibitor (Pharmacia). Following denaturation for 10 min at 65°C, RNA was reverse transcribed for 1 h at 42°C, and then the RT enzyme was inactivated by incubation at 95°C for 5 min.
PCR was performed with an aliquot of 5 µl of synthesized cDNA product in a reaction mixture containing 5 µl cDNA, 5 µl 10x PCR buffer (200 mM Tris-HCl, pH 8.4, and 500 mM KCl), 3 mM MgCl2, 50 pmol A and B oligonucleotide primers, 1 µl 5 mM dNTPs, 2.5 U Taq polymerase, and distilled water to a total volume of 50 µl. The conditions were as follows: 3 min denaturation at 94°C, 5 min annealing at 50°C, and 5 min extension at 72°C for the first cycle, and denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C for the subsequent cycles. The cDNA was then amplified in a DNA minicycler (AECL, Chalk River, Ontario, Canada) at 35 and 25 cycles for IL-15 and actin, respectively. The number of cycles for PCR was selected based on the linearity of the PCR product (data not shown). The cDNA concentrations were normalized to yield equivalent actin PCR products to allow for comparison of IL-15 mRNAs. The cytokine-specific primer pairs, having sequences of IL-15A (5'-ATGAGAATTTCGAAACCACATTTG-3') and IL-15B (5'-CCATTAGAAGACAAACTGTTCTTTGC-3'), ß-actin A (5'-CCTTCCTGGGCATGGAGTCCT-3'), and ß-actin B (5'-GGAGCAATGATCTTGATCTTC-3'), were used (17). An aliquot of PCR product (20 µl) was electrophoresed on a 1% agarose gel and visualized by ethidium-bromide staining. The specificity of the PCR products for IL-15 was confirmed by its predicted size on agarose gels and also by Southern blot analysis.
Southern analysis
Amplified DNA was blotted on a positively charged nylon membrane (Boehringer Mannheim, Indianapolis, IN) overnight at ambient temperature in a 10x SSC (3 M NaCl and 300 mM sodium citrate, pH 7.0) and immobilized by UV cross-linking. The membrane was then prehybridized and hybridized at 58°C using rapid-hyb buffer (Amersham Life Sciences, Arlington Heights, IL) for 4 h and overnight, respectively. The synthesized PCR products were then probed with a 32P-labeled IL-15 probe complementary to sequences recognized by the PCR amplification primers (5'-ATGTCTTCATTTTGGGCTGTTTCAGTGCAG-3') (17). Amplified actin cDNA was detected using 32P-labeled human actin cDNA excised by EcoRI restriction enzyme from the actin containing the 1.1-kb plasmid (Bluescript SK-; ATCC). After hybridization, the blots were washed (15 min/wash) with solutions comprising 0.1% SDS, twice at ambient temperature with 2x SSC and once with 0.1x SSC at 37°C, followed by 0.1x SSC at 58°C. Intensity of the probed products was determined using a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). The amount of IL-15 RNA was quantitated for each sample at each time point relative to its own actin RNA level. The given ratio was compared with the RNA transcripts from mock-infected cells infected as described previously (23). All reactions were conducted in conditions in which amplification was linear.
Statistical analysis
Results are presented as mean ± SE. Statistical significance was determined using a Student’s t test and p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined the NK cytotoxic activity of PBMC following
their infection with different viruses as described in Materials
and Methods. All viruses used (influenza virus, HSV-1, EBV,
reovirus, VSV, Sendai virus, and RSV) were able to significantly
enhance the NK activity of PBMC following 20 h of infection as
compared with the mock-infected PBMC (Fig. 1
). Infection with the influenza virus
induced a 38% increase in NK cytotoxicity as compared with 7%
cytocidal activity of the mock-infected PBMC; these results correspond
to a more than 5.5-fold increase in cytocidal activity. Also, VSV had
an activity of 43%, the highest increase in NK activity among the
different viruses. RSV (15%) and EBV (20%) induced a lower increase
in NK cytotoxicity. To determine the role of IL-15 in the observed
induction of NK cytotoxicity, a mAb to IL-15 was used. It was thus
found that this Ab inhibited the increase in NK activity following
infection with the different viruses. This increase was statistically
significant with a p value of 
0.01 and was seen with all
the viruses to a varying degree. Hence, the role of IL-15 in mediating
the induction of NK activity following infection with influenza virus,
HSV-1, EBV, reovirus, VSV, Sendai virus, and RSV was shown.
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The above observations pointed to a role for IL-15 in the
up-regulation of NK cytotoxicity by the different viruses used. If this
was true, the presence of bioactive IL-15 secreted in the supernatants
of virus-infected cells should be detected. In fact, treatment of PBMC
for 24 h with the supernatants of infected PBMC obtained 20 h
PI showed that all supernatants were able to significantly enhance NK
activity as measured by percentage of cytotoxicity in comparison with
the mock-treated PBMC (Fig. 2). To document
the specificity of NK induction by the supernatants of infected PBMC
cultures via IL-15, a mAb to IL-15 was used. The results show that,
when added to the supernatants, anti-IL-15 Ab inhibited the
increase in NK activity. This abrogation of the increase of NK activity
was statistically significant and observed with all viruses
(p 0.01). A more drastic decrease was seen
with the supernatants of EBV and RSV (i.e. from 18 to 2% and from 24
to 3%, respectively), suggesting that in the case of these two viruses
NK cytotoxicity was solely induced by IL-15. These results provide
strong evidence for the presence of IL-15 in the supernatants of
virus-infected PBMC and its role in the induction of NK cytotoxicity.
Moreover, when CD16-depleted PBMC (following two repeated treatments
with anti-CD16 plus complement) were used in the presence of
rIL-15, no up-regulation of NK cytotoxicity was observed (results not
shown).
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, and IFN-
(Fig. 3). These results clearly showed a
significant decrease in NK activity following the use of anti-IL-15
Ab only, and not with mAbs to other cytokines. This was demonstrated in
individual experiments with each virus (Fig. 3, A–C). Interestingly, previous studies with HHV-6
in which Abs to IFN- were used also showed no decrease in NK
cytotoxicity (17). Taken together, our results clearly
indicate that IL-15 is the cytokine specifically responsible in our
experimental system for the up-regulation of NK activity following
infection of PBMC with different viruses.
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After ascertaining the presence of IL-15 in the supernatants of
infected PBMC using a specific mAb, the next step was to test the
bioactivity of the secreted protein. Attempts to detect IL-15 in the
supernatants of infected PBMC by ELISA were unsuccessful (data not
shown). This is because the detection limits of most commercially
available ELISA kits are probably well above the actual bioactive
protein concentrations present in the supernatants of infected PBMC. In
fact, contrary to other cytokines, the concentration of IL-15 is not
directly correlated with the cellular level of IL-15 mRNA expression
(24, 25). However, we were able to detect bioactive IL-15
levels in the supernatants of infected PBMC 20 h PI using the
CTLL-2 proliferation assay as described in Materials and
Methods (Fig. 4). Indeed, all
supernatants obtained from virus-infected PBMC cultures supported the
growth of the CTLL cell line as illustrated by the significant increase
in [3H]thymidine incorporation (cpm) (treatment
of supernatants with a mAb to IL-2 ascertained that the proliferation
of CTLL-2 was not due to IL-2; data not shown). Interestingly,
supernatants from VSV-treated cells, for example, had a high increase
correlating with their high induction of NK activity, whereas
supernatants from Sendai virus-treated cultures showed lower values,
which correlated with their lower NK-inducing activity (see Fig. 2
for
comparison).
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To determine the levels of IL-15 mRNA expression at different time
intervals (i.e., 2, 4, 8, 12, and 20 h) PI of PBMC, cells were
lysed and mRNA levels assessed by semiquantitative RT-PCR (Fig. 5). IL-15 mRNA levels for most viruses peaked
at 8 h PI, with some variation among the different viruses:
influenza virus, HSV-1, EBV, and Sendai virus peaked at 8 h PI,
whereas reovirus and RSV had little increase between 8 and 12 h PI
(Fig. 5, A and B), and VSV peaked at 4 h PI,
probably accounting for the higher increase observed in NK induction
(Fig. 1). However, there were differences with respect to the
correlation between the increase in mRNA levels and protein expression
levels. For instance, EBV mRNA expression peaked at 8 h PI, with a
40-fold increase in mRNA levels. In comparison, influenza IL-15 mRNA
level also peaked at 8 h PI with a 28-fold increase. For both
viruses, the bioactive protein levels (Fig. 4), as well as the NK
activity (Fig. 1) did not correlate with the observed mRNA levels.
Compared with EBV, influenza virus infection of PBMC was associated
with a higher concentration of IL-15 protein and a much higher NK
activity, thus pointing to the possibility of a differential regulation
at the translational and posttranslational levels of IL-15 expression.
This is also true if one compares other viruses such as HSV-1 and
influenza, which despite different expression levels of mRNA following
infection (10- and 28-fold increase, respectively), induced NK
activity of comparable strength and expressed similar levels of the
bioactive protein (Figs. 1 and 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß IFNs produced by infected cells that up-regulate killing
mechanisms, and IL-12, which promotes IFN- secretion
(28). The role of NK cells in early antiviral immune
defense mechanisms seems to be crucial as evidenced by studies of
several types of viral infections, particularly those due to
herpesviruses (14, 29). Numerous studies have
demonstrated that NK cells can selectively lyse virus-infected target
cells while sparing uninfected cells (30, 31). The
expression of viral Ags or other surface structures by infected cells
appears to render them more sensitive to NK cytolysis
(30, 31, 32). Mammalian cells infected by many different
viruses such as herpes, vaccinia, measles, mumps, and influenza viruses
can be lysed in vitro by NK effectors (30). Furthermore,
several studies have shown that humans and mice with a relatively low
NK activity are more susceptible to herpesvirus infections such as by
HSV, CMV, and EBV (14, 30, 31, 33, 34, 35). Characterization
of the virus-induced endogenous mechanisms regulating NK cell responses
and functions has been limited to the IFN /ß-mediated activation
of NK cell cytotoxicity. However, Binder et al. (36),
using a murine model of viral infection, showed that deletion of the
IFN-/ß receptor gene did not affect NK cell expansion and
activity, thus suggesting the existence of alternative pathways and/or
cytokines that can mediate the enhancement of NK cytotoxicity. Recent
work has shown that some, but not all, viral infections induce IL-12,
the expression of which results in IFN- production by NK cells,
which in turn contributes to an antiviral state (29).
However, a newly identified cytokine, IL-15, with biologic activities
similar to IL-2, including the ability to up-regulate NK cytolytic
mechanisms (4), has been shown to be induced following
infection with two related human lymphotropic herpesviruses, HHV-6 and
HHV-7 (17, 18). A study by Carson et al. (37)
showed that human monocytes produced IL-15 protein within 5 h of
activation with LPS and that it was critical for optimal NK cell
production of IFN-. Furthermore, Elloso et al. (38)
reported that neutralization of endogenous IL-15 in PBMC from
HIV-infected patients, in which dysfunction of both NK and ADCC
activity is well documented (39, 40), resulted in a
reduction in IFN- production in vitro. A previous report had shown
that IL-15 and IL-12 had the ability to enhance cell-mediated immunity
of HIV-infected PBMC (41), and recent work from our
laboratory has shown that adding rIL-15 to PBMC culture from
HIV-infected individuals could up-regulate NK and ADCC activity in
vitro (42). Taken together, these observations suggested
that IL-15 plays an important role in the up-regulation of NK effector
function following viral infection.
To gain further insight into the role of IL-15 in the activation of the
early nonspecific cellular immune response to viral infection, we
studied the induction of IL-15 following exposure of human PBMC to
several viruses belonging to different families (influenza virus,
HSV-1, EBV, reovirus, VSV, Sendai virus, and RSV), and its role in the
enhancement of the NK cytocidal activity of virus-infected PBMC. Our
data clearly show that all the viruses studied induced the expression
of IL-15, both at the mRNA and protein levels, and that these viruses
significantly enhanced the NK activity of PBMC as compared with the
mock-infected cells. This increase in NK cytotoxicity was shown to be
abrogated by the use of a mAb to IL-15. Furthermore, when the
supernatants from virus-treated PBMC cultures were added to normal
PBMC, the NK activity of the latter cells was up-regulated. These data
thus show that NK activity is up-regulated following viral infection
via IL-15 induction. Furthermore, the present results clearly show that
IL-15 was specifically responsible for the NK induction of normal PBMC
following treatment with supernatants of infected PBMC. Inhibition of
NK activity was only seen with anti-IL-15 Abs and not with mAbs
specific to other cytokines such as IL-12, IL-2, IFN-, or TNF-.
Based on these findings, it would appear that IL-15 is secreted earlier
than the other cytokines following a viral infection. This finding is
supported by the fact that monocytes are the primary source of IL-15
production in response to intracellular infection, as documented in
several studies (17, 43, 44, 45). These findings emphasize the
importance of studying the role of IL-15 early in infection as well as
in the production of other cytokines.
It has been difficult to demonstrate IL-15 in the supernatants of the majority of the cells that express messages for this cytokine, despite its widespread expression in several human tissues (25). The results obtained in this study regarding the mRNA and the levels of IL-15 protein expression illustrate the differential regulation of this cytokine. In fact, IL-15 mRNA levels were not translated into equivalent proportions of bioactive protein as assessed by the CTLL-2 bioassay. This could be explained in the light of the translational and posttranslational levels of regulation of IL-15 protein synthesis. Unlike other cytokines that belong to the same family (such as IL-2, which is regulated at the level of message transcription and stabilization), synthesis and secretion of IL-15 appear to be controlled by the presence of upstream AUGs in the 5' untranslated region (UTR) of the IL-15 messages (24, 25). It has been hypothesized that IL-15 is stored in a translationally inactive IL-15 mRNA form that can be readily translated in response to an infection through several mechanisms that are effective in the removal of the 5' UTR blockade of transcription such as a splicing event or an internal initiation of translation (46, 47, 48). Interestingly, Bamford et al. (24) showed by analysis of the IL-15 message from the HuT-102 T cell line, that there was a 6- to 10-fold more protein expressed in HuT-102 cells compared with activated monocytes, correlating with the lack in HuT-102 cells of 8 of the 10 upstream AUGs normally present in the 5' UTR of the IL-15 message. Furthermore, IL-15 secretion was found to be controlled by natural signal peptides that apparently regulate the efficiency of release of soluble IL-15 in biologically relevant amounts (49, 50). Meazza et al. (49) reported that substitution of the natural signal peptide encoded by IL-15 cDNA with another one from a secretory protein IgVx chain (VxL) increased significantly the secretion of biologically active IL-15. Hence, the results obtained in the present study are consistent with other findings, suggesting that IL-15 synthesis and secretion is controlled at multiple levels (translation and entry into the secretory pathway), in addition to transcription (24).
Innate immunity is an important first line of defense and plays a key
role in immune surveillance, particularly against infections.
Unraveling the role of cytokines involved in the regulation of the
early immune response to infections should also lead to a better
understanding of their role in host defense and pathogenesis of these
infections. With respect to IL-15, the results presented confirm its
role in the activation of NK cytocidal activity. In this context, it is
noteworthy that a recent study by Carson et al. (51)
documented the ability of IL-15 to sustain NK cell survival with the NK
cells expressing IL-15R mRNA. It was shown that picomolar amounts of
IL-15 were sufficient, in the absence of serum or other growth factors,
to sustain the survival of resting human NK cells for up to 8 days. It
is thus likely that IL-15 plays a unique role in the activation and
maintenance of the host’s innate cellular immune response to
infection. Indeed, recent data from our laboratory showed that
long-term incubation of IL-15 with EBV-infected PBMC in vitro resulted
in the inhibition of EBV-transformed/immortalized cells in these
cultures (E. Sharif-Askari, L. M. Fawaz, and J. Menezes, in
preparation), which further supports a role for IL-15 in antiviral
immunity. Nevertheless, given that treatment with anti-IL-15 Abs
did not block all the enhanced NK cell cytotoxicity observed with
several of the viruses used in our assay system, it is likely that
other factor(s) (e.g., other cytokines) is (are) contributing to the
up-regulation of NK activity by these viruses. Whether the action of
such factor(s) is synergistic with or complementary to that of IL-15
remains to be addressed in future studies.
In conclusion, the results presented demonstrate that exposure of PBMC to different, unrelated viruses leads to immediate activation of NK cytotoxic effector function via IL-15 induction. These events may indeed represent crucial steps in host’s innate immune response to, and immunosurveillance against, viral infections.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada.
3 Current address: Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
4 Address correspondence and reprint requests to Dr. José Menezes, Laboratory of Immunovirology, Hôpital Sainte-Justine, 3175 Côte Ste-Catherine, Montreal, Quebec, H3T 1C5 Canada. E-mail address:
5 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; HHV, human herpesvirus; VSV, vesicular stomatitis virus; RSV, respiratory syncitial virus; UTR, untranslated region; PI, postinfection.
Received for publication September 17, 1998. Accepted for publication July 28, 1999.
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