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Production by Peripheral Blood Mononuclear Cells in Response to Viral Stimulation1
*
Department of Pathology and Laboratory Medicine, and
Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, however,
has not been reported. In this study, PBMC from healthy donors were
stimulated with virus in the presence of IL-10. Human IL-10 (hIL-10)
caused reductions in both the frequency of IFN--producing cells
(IPC) and bulk IFN in response to herpes simplex virus type-1 (HSV-1),
Sendai virus, Newcastle disease virus, and vesicular stomatitis virus.
The inhibitory effect occurred when IL-10 was added 2 or 4 h
before, or 2 h poststimulation with HSV or Sendai virus, but not
when added 4 h postinduction. Unlike IL-10, IL-4 did not affect
the IFN- response to HSV. However, when PBMC were induced with
Sendai virus, IFN- production was also reduced by IL-4. IL-10
treatment of PBMC resulted in strong reductions in the steady state
levels of both HSV- and Sendai virus-induced IFN-1, -2, and -ß
mRNA as determined by RT-PCR. IFN- production to Sendai virus occurs
predominantly by monocytes, whereas most enveloped viruses stimulate
low frequency "natural IFN-producing cells (NIPC)," which are
thought to be dendritic cells. Peripheral blood dendritic cells were
found to express the IL-10 receptor, suggesting that IL-10 may directly
act on the dendritic IPC. Addition of monoclonal anti-IL-10 to PBMC
resulted in a significant increase in both the frequency of IPC and the
amount of secreted IFN- in response to HSV but not Sendai virus. We
conclude that human IL-10 can serve as both an endogenous and exogenous
regulator of IFN- production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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belongs to
the type 1 family of IFNs, which are all derived from the same
ancestral gene and exert their effects via a common cell surface
receptor (1). IFN- can be induced by a number of agents including
foreign cells, virus-infected cells, tumor cells, bacterial cells, free
virions, viral envelopes, and synthetic double stranded RNA (2).
Several laboratories, including our own, have been studying the cells
responsible for the secretion of IFN- in response to a number of
different viruses (3). These investigations have demonstrated two major
IFN--producing cell populations: monocytes and a second population
of cells that have been called "natural IFN-producing cells"
(NIPC).3 The NIPC, which
are negative for lineage-specific cell surface markers and share the
phenotype of immature DCs, have been shown to be the primary cells
among PBMC that produce IFN- in response to a number of both
enveloped DNA and RNA viruses (4, 5, 6). Of these, herpes simplex virus
type-1 (HSV-1) and Sendai virus have been the best studied and induce
predominantly NIPC and monocytes to produce IFN-, respectively.
Cederblad et al. have reported that the cytokines GM-CSF and IL-3 are
able to up-regulate IFN- secretion from both monocytes and NIPC in
response to HSV (7). Although there have been several studies
demonstrating the down-regulation of IFN- in various diseases
(8, 9, 10), there is very little information about the factors involved in
the reduction of IFN- production in the patient populations.
IL-10, a pleiotropic cytokine, is a natural immunosuppressant that
shifts the body’s immune response away from an inflammatory response
(11, 12, 13). It is produced by human CD4+ T cells and clones,
monocytes, macrophages, keratinocytes, activated B cells, and B
lymphoma lines and plays a major role in T helper cell regulation,
serving to down-regulate Th1 responses (14, 15). The production of
IL-10 by macrophages can be inhibited by IL-10 itself, which suggests
an autocrine effect of IL-10 on monocyte function (16). In vitro, IL-10
has been shown to inhibit the production of several proinflammatory
cytokines including IL-1ß, IL-6, IL-8, TNF-, GM-CSF, and G-CSF, as
well as to inhibit monocyte MHC class II expression (17, 18). IL-10
also has been shown to synergize with other cytokines, including IL-4
and TGF-ß, to inhibit macrophage cytotoxic activity (19). In addition
to its inhibitory effects on monocyte function, IL-10 has been shown to
exert a wide array of immuno-stimulatory effects on B cells and T cells
(13). Clerici et al. have shown significant increases in the levels of
IL-10 produced by PBMC from HIV infected individuals in whom T helper
cells functions were severely compromised (20). It has been suggested
that sustained production of IL-10 in various organs serves to protect
patients with septic peritonitis, and IL-10 knock-out animals
demonstrate chronic enterocolitis, indicating a failure to regulate
response to bacterial gut pathogens (21). In general, the production of
IL-10 is correlated with expression of Th2-like responses induced by
infectious agents (22).
To determine the effects of IL-10 on IFN- production, we stimulated
PBMC with different viruses including HSV, Sendai virus, vesicular
stomatitis virus (VSV), and Newcastle disease virus (NDV) in the
presence or absence of exogenous hIL-10. The frequency of IPC and the
amount of secreted IFN- were measured by ELISPOT and IFN bioassay,
respectively. The influence of endogenously produced IL-10 on IFN-
secretion by PBMC stimulated with HSV and Sendai virus was also
investigated. We observed that hIL-10 inhibited both the frequency of
IPC and bulk IFN- as well as the steady state levels of IFN-
mRNA. Furthermore, blocking of endogenous IL-10 enhanced both the
frequency of IPC and the amount of secreted IFN- produced in
response to HSV but not Sendai virus, suggesting a role for endogenous
IL-10 in regulation of IFN- synthesis in certain viral
infections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GM-0459A (Human Genetic Mutant Cell Repository, Camden, NJ), a human fibroblast cell line trisomic for chromosome 21, was grown in DMEM (JRH, St. Lenexa, KS) supplemented with 15% FCS (Gemini Bio-Products, Inc., Calabasas, CA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (DMEM-15% FCS). The cells were passaged twice weekly and used up to passage 20. Vero cells (originally obtained from the American Type Culture Collection (ATCC, Manassas, VA) were grown in DMEM-10% FCS.
Cytokines and Abs
Recombinant IL-4 was purchased from R&D Systems (Minneapolis, MN). Recombinant hIL-10 and anti-hIL-10 mAb (JES3) were kindly supplied by Dr. Sidney Smith, (Schering-Plough, Kenilworth, NJ). Rat IgG (isotype control for JES3) was purchased from Sigma (St. Louis, MO). Fluorescent-conjugated mouse anti-human CD14 and its isotype control were obtained from DAKO Corporation (Carpinteria, CA).
Viruses
HSV-1 strain 2931 was grown and titered on Vero cells as previously described (23). VSV, originally obtained from Dr. Nicholas Ponzio of New Jersey Medical School, was also grown and titered on Vero cells. Sendai virus, strain Sendai/Cantell, was obtained from ATCC and grown in 10-day-old embryonated chicken eggs. NDV, strain VR-107, was also grown in embryonated chicken eggs. Sendai virus and NDV were titered in a hemagglutination assay using chicken RBC, and titers were expressed as hemagglutination units (HAU)/ml. All viruses were stored at -70°C.
Cell preparation
PBMC were prepared by Ficoll-hypaque (Lymphoprep; Accurate Chemical and Scientific, Westbury, NY) density sedimentation of heparinized peripheral blood obtained with informed consent from healthy volunteers. The cells were washed twice in HBSS with Ca2+/Mg2+ (Mediatech, Herndon, VA) and resuspended in RPMI 1640 (JRH Biosciences) supplemented with 10% FCS, penicillin, streptomycin, L-glutamine, and 25 mM HEPES buffer (RPMI-10%). Cells were enumerated using a coulter Model ZBI electronic particle counter (Coulter Electronics, Hialeah, FL).
For the enrichment of CD14+and CD14- populations, magnetic cell separation was used. PBMC were incubated with mouse-anti-human CD14 Ab for 1 h at 4°C, washed, and resuspended in magnetic beads conjugated with sheep anti-mouse IgG and incubated at 4°C for another hour followed by separation with the MACS magnet (Miltenyi Biotec, Sunnyvale, CA). Both CD14+ and CD14- were harvested, and the purity of the populations was assessed by flow cytometry.
Analysis of IL-10 receptor expression on DCs
The expression of IL-10 receptor was determined for whole PBMC using an IL-10 Fluorkine Kit (R&D Systems) according to the manufacturer’s protocol. PBMC (1 x 106) were first stained with Tricolor-labeled anti-HLA-DR (Caltag, Burlingame, CA) and PE-conjugated anti-CD3, CD14, CD19 (DAKO) and CD16 and CD20 (both from Becton Dickinson, Sunnyvale, CA). The cells were then washed and separated into two groups to which were added the biotin-conjugated IL-10 or the biotin-labeled control protein. After incubation, avidin-FITC was added, followed by washing. Groups containing appropriate isotype controls were also used for each fluorochrome. All tubes were then washed and fixed in 1% paraformaldehyde for flow analysis. Analysis was conducted on a FACSCaliber (Becton Dickinson). The PBMC were first analyzed for forward and side scatter, and the cells within the lymphocyte and monocyte gate were acquired. These were then analyzed for the tricolor and PE markers, and cells that were HLA-DR+ but negative for the PE-conjugated Abs were further analyzed for FITC-IL-10 binding.
ELISPOT assays
The ELISPOT assay for determination of frequency of IPC was
performed as previously described (9). PBMC in RPMI-10% FCS were
incubated at 1 x 106 cells/ml with viruses at
concentrations as described in the experiments for 6 to 8 h at
37°C in a 5% CO2 incubator. Microtiter plates (96-well)
with nitrocellulose membrane bottoms, type HATF (Millititer HA plates,
Millipore, Bedford, MA), were coated with ammonium sulfate-precipitated
bovine anti-human IFN- antiserum (Batch No. AS/46/93, kindly
provided by Wellcome Research Foundation, Beckenham, U.K.) and then
fixed with 0.05% glutaraldehyde. Stimulated or mock-stimulated PBMC
were washed and then added in serial threefold dilutions to the
Millititer plates and to standard 96-well flat-bottom plates (Corning,
Acton, MA) for bulk IFN generation. Cells in the Millititer plates were
incubated at 37°C for 10 to 12 h to allow production and capture
of IFN- and were developed using a murine anti-human IFN- mAb
that cross-reacts with different subtypes of IFN- (293, kindly
provided by Drs. Brita Cederblad and Gunnar Alm, Uppsala, Sweden),
followed by horseradish peroxidase-conjugated goat anti-mouse IgG
(Jackson Immunoresearch Laboratory, West Grove, PA) and the horseradish
peroxidase substrate 3,3' diaminobenzidine tetrahydrochloride (Sigma).
The resulting brown spots were counted under a dissecting microscope,
and the frequencies of IPC were calculated and expressed as mean
IPC/104 PBMC. The SDs for replicate wells were typically
less than or equal to 10% of the mean values.
IFN bioassays
IFN bioassays were performed on samples harvested from the
standard microtiter plates described above. Supernatants were stored
frozen at -20°C before quantification of IFN activity. IFN was
quantified using a cytopathic effect (CPE) reduction assay as
previously described (24) using GM 04592A as the indicator cell line
and VSV as the challenge virus. Each IFN assay contained cell and virus
controls, and the international leukocyte IFN (IFN-) reference
standard G-023-901-527, obtained from the National Institute of Allergy
and Infectious Diseases. The lowest detectable IFN concentration was
between 3 and 10 IU/ml of supernatant.
RNA isolation and RT-PCR analysis
Total RNA was prepared from virally stimulated PBMC using RNAzol
B according to the manufacturer’s protocol (TEL-Test, Friendswood,
TX). Reverse transcription and PCR were performed using the
Perkin-Elmer Cetus Gene Amp RNA PCR kit (Perkin-Elmer Cetus, Norwalk,
CT) using 1 µg of RNA from 107 PBMC. All primers
were synthesized by Operon Technologies (Alameda, CA): IFN-1 sense
primer, 5'-CAATATCTACGATGGCCTCGC-3'; IFN-1 antisense primer,
5'-AGAGATGGCTGGAGCCTTCTG-3' (25); IFN-2 sense primer,
5'-ATCCAGCAGATCTTCAATCT-3'; IFN-2 antisense primer,
5'-AAGAAAAAGATCTCATGATT-3'; IFN-ß sense primer,
5'-GATTCATCTAGCACTGGCTGG3'; IFN-ß antisense primer,
5'-CTTCAGGTAATGCAGAATCC-3' (25); ß-actin sense primer,
5'-GTGGGGCGCCCCAGGCACCA-3'; and ß-actin antisense primer,
5'-GTCCTTAATGTACGCACGATTTC-3' (16). For IFN-1 and IFN-ß PCR, the
products were denatured at 94°C for 45 s, annealed at 6°C for
45 s, and extended at 72°C for 2 min. For IFN-2 PCR, the
products were held at 95°C for 2 min, annealed at 55°C for 1 min,
extended at 72°C for 1 min, and denatured at 95°C for 1 min.
ß-actin PCR was performed using both sets of conditions. A total of
26 cycles of amplification were performed using the Gene Amp PCR system
9600 (Perkin-Elmer). After the PCR amplification reaction, 20 µl of
each reaction was loaded on 2% agarose gels in TBE buffer, and PCR
products were quantitated using a Fluorimager SI (Molecular Dynamics,
Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and frequency of NIPC in
response to viral stimulation
PBMC from healthy individuals were stimulated for 6 h with
either HSV at a multiplicity of infection (MOI) of 1 or Sendai virus at
16 HAU/ml in the presence or absence of different concentrations of
hIL-10. The treated cells were placed into Millititer plates or
flat-bottom plates for an additional 12 h for ELISPOT and bulk IFN
assays, respectively. IL-10 inhibited both the frequency of IPC as well
as total IFN produced in response to both HSV and Sendai virus in a
dose-dependent manner (Fig. 1
). In
addition to decreasing the frequency of detected IPC, IL-10-treated
PBMC also tended to yield fainter spots in the ELISPOT assay, which is
interpreted to indicate that, on a per cell basis, the IL-10 decreases
IFN- production (data not shown). The inhibition was maximal at 10
ng/ml IL-10, and increasing amounts of IL-10 did not further enhance
the inhibition. Therefore, a concentration of 10 ng/ml IL-10 was chosen
for additional experiments. Statistical analysis using nine individual
donors indicated that IL-10 treatment resulted in 2.3- and 2-fold
reductions in frequencies of IPC responding to HSV and Sendai virus,
respectively, as well as 4.6- and 3.3-fold reductions in bulk IFN
production, all of which were highly significant (data not shown.)
Moreover, inclusion of monoclonal anti-IL-10 Ab (10 µg/ml)
totally abrogated the inhibitory effects of exogenously added IL-10
(Fig. 2), confirming the specificity of
the inhibitory effects of IL-10 on IFN- production.
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response from PBMC (26). We therefore tested the effects of
IL-10 on the frequency of NIPC and IFN- secreted by PBMC that were
stimulated with either NDV or VSV, two RNA viruses. IL-10 inhibited the
frequency of NIPC 65% and 55% and bulk IFN- 6.5-fold and 3-fold
from PBMC stimulated with NDV or VSV, respectively (Fig. 3).
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when added up to 2 h
postinduction with HSV or Sendai virus
To further define the parameters of IL-10 inhibition of IFN-
production, IL-10 (10 ng/ml) was added to PBMC culture at different
times before and after the start of stimulation by either HSV or Sendai
virus. As shown in Figure 4, hIL-10
inhibited the frequency of NIPC and the amount of IFN- secreted by
PBMC stimulated with either HSV or Sendai virus when added either
2 h before or 2 h after addition of viruses. However, when
IL-10 was added 4 h after addition of viruses, no inhibition was
observed. These results indicate that IL-10 acts during early phases of
IFN- induction.
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production
In addition to IL-10, other cytokines including IL-4 have been
shown to be important antiinflammatory agents that suppress
proinflammatory activities of macrophages by down-regulating the
production of TNF-, GM-CSF, G-CSF, IL-6, and IL-8 (19, 27, 28, 29). We
therefore investigated the effect of IL-4 on production of IFN- by
PBMC stimulated with virus. IL-10 but not IL-4 significantly lowered
the level of secreted IFN- and the frequency of NIPC responding to
HSV (Table I). Different concentrations
of IL-4 (from 0.1 to 10 ng/ml) or up to a 4-h preincubation with this
cytokine did not lower the level of IFN- secreted by PBMC stimulated
with HSV (data not shown).
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-producing cells and bulk IFN- secreted by PBMC stimulated
with Sendai virus. The data for inhibition of Sendai virus-induced but
not HSV-induced IFN- by IL-4 is in agreement with published data by
Gobl et al. (30). No effects on HSV- or Sendai virus-induced IFN were
observed using TGF-ß or IL-13 (data not shown).
IL-10 inhibits IFN-1, -2, and -ß at the mRNA level
Several reports have demonstrated that IL-10 inhibition of
cytokine production is accompanied by decreased steady-state levels of
mRNA (31, 32). IFN-1, -2, and -ß mRNA are known to be strongly
expressed by NIPC and monocytes in response to viral stimulation (33).
We therefore investigated the effects of IL-10 on the mRNA levels for
IFN-1, -2, and -ß in PBMC stimulated by either HSV or Sendai
virus. Total RNA was prepared as described in Materials and
Methods, reverse transcribed into cDNA, and subsequently amplified
with cytokine-specific primers and run on 2% agarose gels with
ethidium bromide.
PBMC stimulated with either HSV or Sendai virus resulted in strong
IFN- and -ß mRNA expression whereas the addition of IL-10 with
virus inhibited these mRNA levels (Fig. 5). The levels of inhibition of IFN-
specific mRNA correlated well with inhibition of both frequency of IPC
and secretion of IFN- by PBMC stimulated with either HSV and Sendai
virus.
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production by PBMC in response to HSV stimulation
Van der Poll et al. (34) have proposed that endogenous IL-10
protects mice from death during septic peritonitis. They were able to
demonstrate the elevation of TNF levels in serum of mice pretreated
with anti-IL-10. Clerici et al. (20) have found significant
increases in the levels of IL-10 production by PBMC from
HIV+ individuals in whom Th cell functions were
severely compromised. In an in vitro study, they demonstrated that
neutralization of IL-10 reversed the defect in Ag-specific Th function
from HIV-infected individuals. To evaluate the role of endogenous IL-10
in the regulation of IFN- production, PBMC were stimulated with HSV
and Sendai virus in the presence or absence of anti-IL-10, and the
frequency of NIPC and bulk IFN generation were determined. Anti-IL-10
at a concentration of 20 µg/ml significantly increased the frequency
of NIPC and the amount of IFN- production by HSV stimulated PBMC
whereas isotype control Ab had no effect (Fig. 6A). Anti-IL-10,
however, did not have a significant effect on IFN- production by
PBMC induced with Sendai virus (Fig. 6B).
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production by both NIPC and monocytes
Human monocytes are important producers of several cytokines,
including IL-1, IL-6, IL-8, TNF-, and GM-CSF. Several
laboratories, including ours, have shown that, in addition to NIPC,
monocytes are the second major population in peripheral blood that
produce IFN- (3, 5). IL-10 has been shown to inhibit the production
of IL-1, IL-6, and IL-8 by LPS-activated human monocytes. To
determine the effects of IL-10 on the production of IFN- by highly
purified monocytes (CD14+) vs CD14-
cells, we separated these populations using a magnetic microbead
separation system. Each population was stimulated with either HSV or
Sendai virus in the presence or absence of hIL-10, and the frequency of
IPC and the amount of secreted IFN- were determined. IL-10 inhibited
the frequency of IFN--producing cells from both CD14+
and CD14- populations as well as bulk IFN- secreted by
these cells (Table II). The
CD14+ cells failed to produce IFN- in response to HSV
but responded well to Sendai virus stimulation. Since the monocytes
were very pure (97% CD14+), this suggests that the IL-10
may be acting directly on this population to suppress Sendai
virus-induced IFN- production. A direct effect of IL-10 on NIPC in
the CD14- population cannot be inferred from this data
since the NIPC represent only a small fraction of the
CD14- cells. Therefore, studies were undertaken to
determine whether peripheral DCs, to which the NIPC belong, express the
IL-10 receptor.
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Recently, human and mouse IL-10 receptors have been identified and
characterized on several cell lines, including human B cell, T cell,
monocytic, promonocytic, and NK cell lines (35, 36, 37, 38). Cross-linking
studies have indicated a molecular size of 90 to 120 kDa for the hIL-10
receptor, with a few hundred receptors detected per cell with
affinities in the 50 to 250 pm range. However, the presence of hIL-10
receptor on human peripheral blood DC has not been reported. We
therefore used biotinylated IL-10 followed by avidin-FITC to detect the
presence of IL-10 receptor on DC. DC were identified among PBMC by
their expression of HLA-DR but failure to express CD3, CD14, CD16,
CD19, or CD20 (Fig. 7B). Of the cells
identified as DC (region R1 in Fig. 7B), 90% were
found to bind IL-10 and therefore express the IL-10 receptor (Fig. 7C). These IL-10 receptor positive cells had a
forward and side scatter typical of DC (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by PBMC stimulated
with either HSV or Sendai virus. The inhibition of IFN- by IL-10 was
dose dependent and reversed by addition of a neutralizing
anti-IL-10 mAb, illustrating the specificity of IL-10 effects. The
inhibitory effect of IL-10 was partial, even at saturating
concentrations, and ranged from 60% to 85%. IL-10 also inhibited the
frequency of NIPC and bulk IFN- production by PBMC that were
stimulated with two other viruses, NDV and VSV. It remains to be
determined whether the observed IL-10 effects are direct or mediated
via different factors, indirectly. However, the presence of the IL-10
receptor on both monocytes (38) and peripheral blood DCs, the kinetics
of inhibition of IFN- production, and the IL-10 suppression of
IFN- production in response to Sendai virus in highly purified
monocytes all suggest that the IL-10 acts directly on the
IFN--producing cells.
Time course studies demonstrated that hIL-10 added at 4 h and
2 h prior or 2 h poststimulation but not after 4 h
postinduction, inhibited the IFN- production by PBMC stimulated with
either HSV or Sendai virus. However, pretreatment of the cells with
IL-10 did not enhance the inhibitory effect of IL-10, whereas PBMC
stimulated with viruses for 4 h were subsequently resistant to the
suppressive effects of IL-10. These results demonstrate that rIL-10
affects an early step of IFN- induction. These kinetics are
consistent with those reported for the inhibitory effects of IL-10 on
the production of the proinflammatory cytokines IL-1ß, IL-6, and
TNF- by LPS-stimulated PBMC (39).
IL-4 shares many common effects with IL-10 on macrophages. Oswald et
al. have shown the synergistic effect of IL-10 with IL-4 and TGF-ß to
inhibit macrophage cytotoxic activity (19). Hart et al. have shown that
IL-10 and IL-4 inhibit the production of IL-1ß by synovial fluid
macrophages and blood monocytes from patients with inflammatory
arthritis (31). These observations prompted us to study whether IL-4,
similar to IL-10, has an effect on IFN- production by PBMC in
response to HSV or Sendai virus. In PBMC from healthy donors, we have
confirmed the work of Gobl et al., who demonstrated that IFN-
produced in response to Sendai virus but not HSV is inhibited by IL-4
(30). In contrast to the selective inhibition by IL-4, IL-10
significantly inhibited IFN- produced by PBMC in response to either
HSV or Sendai virus, suggesting unique pathways for these inhibitory
cytokines. Moreover, neither TGF-ß nor IL-13 affected IFN-
production in response to either stimulus. Several laboratories,
including our own, have shown the presence of two major
IFN--producing cell populations among PBMC: the dendritic NIPC and
monocytes (5, 6, 26). The early events in the production of IFN- by
NIPC and monocytes in response to HSV and Sendai virus are very
different. In addition to Sendai virus inducing a more frequent
monocytic IPC population, this IFN- production is insensitive to
inhibition with the lysosomotropic drug, chloroquine, whereas
production of IFN- by NIPC in response to a number of viruses,
including HSV, is clearly dependent on endosomal acidification (Ref.
40; and M. Milone and P. Fitzgerald-Bocarsly, manuscript in
preparation). Moreover, several lines of evidence have implicated the
mannose receptor as important in recognition and probably uptake of
virus into endosomal compartments by the NIPC but not by the
monocyte.4 Since IL-10
affects IFN production by both the monocytes and NIPC, it seems likely
that this inhibition may affect events downstream of the initial
recognition of viruses by the IPC. Our previous observations of
signaling events during IFN- induction have shown that protein
kinases C and A and tyrosine kinase activity are involved in the
regulation of IFN- production in response to HSV-1 (33). Therefore,
the effect of IL-10 on these kinase activities will be the target of
future investigations in our laboratory.
The mechanism by which IL-10 inhibits the production of other cytokines
by LPS-activated monocytes has been shown to be via degradation of
cytokine mRNA and at the transcription level (41). The work by Wang et
al. has demonstrated that hIL-10 inhibits expression of IL-1ß, IL-6,
and TNF- by acting mainly at the level of cytokine gene
transcription (18). Using RT-PCR, we have demonstrated that hIL-10
inhibits the steady state mRNA levels for IFN-1, -2 and -ß in
PBMC induced either with HSV or Sendai virus. However, nuclear run-on
transcription assays and stability assays will be required to determine
whether hIL-10 inhibits transcription of the IFN- and IFN-ß genes
or effects the stability or processing of the mRNA involved. In
preliminary studies we have found that IL-10 inhibited the elevated
level of NF-
B in monocytes stimulated with Sendai virus (our
unpublished results). Since at least the IFN-ß promoter contains
NF-B binding sites, this observation clearly warrants further
investigation.
Roles for endogenously produced IL-10 in host defense have previously
been reported. Endogenous IL-10 was found to protect mice from death in
a septic peritonitis model (34). Recent data also suggest that the loss
of T helper cell function in HIV+ individuals results, at
least in part, from changes in immunoregulatory cytokine profiles. It
was observed that IL-10 was up-regulated at the mRNA and protein level
by PBMC from HIV+ individuals and that addition of
anti-IL-10 reversed the defective Ag-specific Th cell function (20, 42). We investigated the effect of endogenous IL-10 on IFN-
production by PBMC stimulated with HSV and Sendai virus. Neutralizing
Ab to IL-10 significantly increased the frequency of NIPC and the
amount of IFN- produced by PBMC stimulated with HSV, which suggests
a role for endogenous IL-10 in the regulation of IFN- production in
NIPC. Contrary to its effect on HSV-treated PBMC, anti-IL-10 did
not affect the level of IFN- production by PBMC in response to
Sendai virus. We have found that HSV induces IL-10 message and protein
at higher levels than those induced by Sendai virus (F. Payvandi and P.
Bocarsly, manuscript in preparation). Thus, the differential effects of
endogenous IL-10 may be explained by response of PBMC to these viruses
and suggest an important role for endogenous IL-10 regulation of NIPC.
This induction of IL-10 by virus is also likely to play a major role in
down-regulating IL-12 and TNF- synthesis in addition to IFN-. In
the absence of regulators such as IL-10, secretion of these cytokines
might lead to excessive IFN-
synthesis by NK cells and T cells,
which, in turn, could activate macrophages and lead to host pathology.
These predictions are supported by the previous observations that mRNA
levels for IL-1ß, IL-12, TNF-, and IFN- in the lung tissue of
Toxoplasma-infected IL-10-deficient animals were severalfold
higher than control animals, which suggests a role for endogenous IL-10
in down-regulating cytokine responses to acute intracellular infection,
thereby preventing host pathogenesis (43).
Further, inhibition of IFN- production by IL-10 may play a role in
the regulation of the natural immune response since IFN- has been
found to stimulate NK cell activation and IFN- production (44, 45, 46).
IFN- is proposed to be important for switching Th cells toward of
the Th1 cytokine profile (IL-2 and IFN-) and has been suggested to
enhance cell-mediated immunity (45, 47). On the basis of these
observations, it is reasonable to hypothesize that inhibition of
IFN- production by IL-10 could contribute to changing the cytokine
profile from Th1 to Th2 and away from cell-mediated immunity.
In conclusion, hIL-10 decreases the number of IFN--producing cells
and the amount of IFN- produced by PBMC stimulated by viruses,
including HSV, Sendai virus, VSV, and NDV. This inhibition is specific
for IL-10, since it can be blocked by neutralizing Ab. IL-10 also
inhibits IFN- by both major populations of IFN-producing cells,
monocytes, and NIPC. Furthermore, endogenous IL-10 also plays a role in
inhibition of IFN- produced by PBMC stimulated with HSV, suggesting
that IL-10 may be an important physiologic regulator of IFN production
by the dendritic NIPC.
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Acknowledgments |
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antiserum, Drs. Brita Cederblad
and Gunnar Alm of Uppsala, Sweden for monoclonal anti-IFN- Abs,
Martin Feuerman for statistical work, Dana Stein and Zenaida Garcia for
help with flow cytometry, and Michael Milone for valuable comments. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Patricia Fitzgerald-Bocarsly, Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. E-mail address:
3 Abbreviations used in this paper: NIPC, natural IFN-producing cell; IPC, IFN-producing cell; HSV, herpes simplex virus; NDV, Newcastle disease virus; VSV, vesicular stomatitis virus; DC, dendritic cell; HAU, hemagglutination units; GM-CSF, granulocyte macrophage-CSF; PE, phycoerythrin; MOI, multiplicity of infection.
4 M. Milone, and P. Fitzgerald-Bocarsly. The mannose receptor mediates induction of IFN- in peripheral blood dendritic cells by enveloped DNA and RNA viruses. Submitted for publication.
Received for publication February 14, 1997. Accepted for publication February 18, 1998.
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