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in Peripheral Blood Dendritic Cells by Enveloped RNA and DNA Viruses1
*
University of Medicine and Dentistry of New Jersey–New Jersey Medical School and
The Graduate School of Biomedical Sciences, Newark, NJ 07103
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in response
to challenge by many enveloped viruses including herpes simplex virus
(HSV) and HIV, whereas Sendai virus predominantly stimulates IFN-
production by monocytes. Glycosylated viral envelope proteins are known
to be important for the induction of IFN-. In this study we
demonstrate that stimulation of IFN- synthesis by HSV is inhibited
by a number of monosaccharides, including fucose,
N-acetylglucosamine, and
N-acetylgalactosamine as well as the yeast
polysaccharide mannan, supporting a role for lectin(s) in the IFN-
stimulation pathway. Furthermore, antiserum to the mannose receptor
(MR) also inhibited HSV, vesicular stomatitis virus, and HIV-induced
IFN- production, but failed to inhibit the IFN- induced by Sendai
virus. We further demonstrated that freshly isolated blood DC and
IFN--producing cells responding to HSV stimulation express the MR.
This study therefore implicates the MR as an important receptor for the
nonspecific recognition of enveloped viruses by DC and the subsequent
stimulation of IFN- production by these viruses. Thus, the MR
probably serves as a critical link between innate and adaptive immunity
to viruses, especially given the role of the MR in Ag capture by DC and
the importance of IFN- in shaping immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß, play an important and indispensable role
in resistance to viral infection (1, 2). These important mediators are
produced by cells in response to stimulation with numerous agents,
including viruses, bacteria, synthetic nucleic acids (e.g.,
polyinosinic acid:polycytidylic acid), and small molecules such as the
antiviral compound, imiquimod. Monocytes produce IFN upon infection
with the paramyxovirus, Sendai virus, as well as after incubation with
polyinosinic acid:polycytidylic acid (3, 4, 5). In contrast, most
enveloped viruses, including herpesviruses (e.g., herpes simplex virus
type 1 (HSV-1),3 CMV,
retroviruses (e.g., HIV-1), and rhabdoviruses (e.g., vesicular
stomatitis virus (VSV)), stimulate IFN- production primarily by rare
(
1/1000) cells in peripheral blood that are negative for
lineage-specific markers of T cells, B cells, NK cells, and monocytes
(4, 6, 7, 8). These cells, which have been termed natural
IFN--producing cells, or NIPC, copurify with peripheral blood
dendritic cells (DC) using FACS (9). Most recently, Cederblad et al.
demonstrated using intracellular flow cytometry for IFN- that these
IFN--producing cells (IPC) appear identical to immature DC (10).
Several studies have sought to define the mechanisms involved in the
stimulation of IFN- expression in PBMC by enveloped viruses. Lebon
reported that Abs to HSV-1 glycoprotein D inhibit the induction of
IFN-, and we observed that sera from HSV seropositive, but not
seronegative, donors inhibited IFN production induced by this virus,
suggesting that interaction of HSV-1 envelope proteins with IPC is
necessary to stimulate IFN- synthesis (11, 12). In addition,
chloroquine, a lysosomotropic agent, inhibited the induction of
IFN-, suggesting that a receptor that recycles through the
lysosomal/endosomal compartment may be involved in the induction
mechanism (4, 12). Besides live HSV, UV-irradiated HSV and
HSV-infected, glutaraldehyde-fixed fibroblasts are potent IFN inducers
of IFN- (11, 13, 14). Furthermore, liposomes derived from Sendai
virus envelope are reported to stimulate IFN- production, and
soluble, recombinant HIV-1 gp120 has also been reported to induce
IFN- (15, 16). Although other laboratories, including our own, have
not confirmed the results of Capobianchi that soluble recombinant gp120
is able to induce high levels of IFN- production (in our hands gp120
is a very weak inducer), we have been able to block the induction of
IFN- by PBMC in response to HIV-1-infected H9 cells by either Ab to
CD4 or gp120, thus suggesting a role for the viral glycoprotein in
induction of IFN (Bocarsly et al., unpublished studies). Similar
results were reported by Francis and Meltzer (17).
Although envelope glycoproteins seem necessary for induction of IFN-
by viruses, the nature of their interaction with the IPC is unknown.
Studies by Charley et al. using the porcine coronavirus, transmissible
gastroenteritis virus, however, indicate an important role for
glycosylation of envelope proteins. Transmissible gastroenteritis virus
treated with glycosidases or mutated at N-linked
glycosylation sites of glycoprotein M lost its IFN--inducing
potential (18, 19). This requirement for properly glycosylated viral
envelope glycoprotein lead us to postulate that a lectin receptor on DC
may be involved in the virus-IPC interaction that leads to the
induction of IFN- expression.
Mononuclear phagocytes express numerous receptors with carbohydrate
binding ability. One of the best characterized of these receptors, the
mannose receptor (MR), was initially identified on alveolar macrophages
(20). Subsequently, this receptor was also observed on many
tissue-differentiated macrophages, but it is not expressed on freshly
isolated peripheral blood monocytes (21). The MR binds several
monosaccharides, including fucose, N-acetylglucosamine, and
mannose, with high affinity (20, 22). Several studies have shown that
this receptor has many functions: it internalizes ligands via
receptor-mediated endocytosis, transduces a glycoprotein-induced
mitogenic signal to alveolar smooth muscle cells, and may mediate the
induction of TNF- produc-tion, lysosomal enzyme secretion, and
cytotoxicity by macrophages (20, 21).
In addition to the MR, a galactose/N-acetylgalactosamine binding receptor has been cloned and found to be expressed by rat, murine, and, most recently, human macrophages (23, 24). A murine receptor with similar binding specificity and sequence homology to the MR, DEC205, has also been cloned, and this receptor appears to be expressed primarily on murine DC (25). Lanzavecchia et al. have reported the expression of the MR on monocyte-derived cultured human DC (26). They showed that this receptor plays an important role in Ag capture and transport of this captured Ag to the lysosomal/endosomal compartment for degradation and association with MHC class II. Recently, Noorman et al. demonstrated MR expression on branched DC in the dermis, but not on Langerhans cells in the epidermis (27). They suggest that MR expression may therefore be specific to immature DC and tissue macrophages. Whether peripheral blood DC express the MR, among which are cells with a more immature phenotype that corresponds to the phenotype of the NIPC, has not been reported.
To examine the role of a lectin on DC in the stimulation of IFN-
synthesis, we first assessed the ability of monosaccharides and mannan
to inhibit viral stimulation of IFN production in mononuclear cells.
Since the MR is reported to be on culture-derived DC, we also examined
the ability of a polyclonal anti-MR antiserum to block the IFN
synthesis induced by several viruses, and we assessed MR expression on
freshly isolated DC. This study demonstrates that the MR is expressed
by peripheral blood DC that produce IFN-, and it plays a significant
role in the induction of IFN- synthesis by different viruses. The
inhibition pattern of the monosaccharides examined also indicates that
an additional receptor(s) with a
galactose/N-acetylgalactosamine specificity may also be
involved in this IFN induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D-Glucose (Glc), D-galactose (Gal),
D-mannose (Man), N-acetylglucosamine (Glc-N-Ac),
N-acetylgalactosamine (Gal-N-Ac), L-fucose
(Fuc), and mannan (isolated from Saccharomyces cerevisiae)
were purchased from Sigma (St. Louis, MO) and used at the
concentrations indicated in the experiments. The goat anti-human MR
antiserum (-MR) was a gift from Dr. Philip Stahl (Washington
University, St. Louis, MO). The normal goat serum (NGS) control was
obtained from Atlanta Biologics (Norcross, GA). The -MR and NGS were
used at the dilutions indicated in the experiments. Phycoerythrin
(PE)-conjugated mAb to the MR was obtained from PharMingen (San Diego,
CA).
Viruses
HSV-1 strain 2931 and VSV (originally obtained from Dr. Nicholas Ponzio of New Jersey Medical School) were grown and titrated by plaque forming assay on Vero cells as previously described (28). Sendai virus, strain Sendai/Cantell, was obtained from the American Type Culture Collection (Manassas, VA), grown in 10-day-old embryonated chicken eggs, and titrated by hemagglutination assay using chicken RBC. Allantoic fluid was used in all IFN inductions using Sendai virus. All virus stocks were stored at -70°C until use.
Cell lines
GM-0459A (National Institute of General Medical Sciences Human Genetic Mutant Cell Line Repository, Camden, NJ), a primary fibroblast cell line trisomic for chromosome 21, were grown in DMEM (JRH Biosciences, Lenexa, KS) supplemented with 15% FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (DMEM, 15%). Vero cells (originally obtained from American Type Culture Collection) were grown in DMEM-10% FCS. H9 cells persistently infected with HIV-1 strain IIIB (HTLV-IIIB/H9; obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health (Bethesda, MD), from Dr. Robert Gallo) were grown in RPMI 1640 (JRH Biosciences) supplemented with 10% FCS, L-glutamate, penicillin, and streptomycin (RPMI, 10%).
ELISPOT assays
ELISPOT assays, used to determine the frequency of IPC, were
performed as previously described (29, 30). Briefly, PBMC were isolated
by Ficoll-Hypaque density centrifugation of heparinized venous blood
obtained from healthy volunteers with informed consent. PBMC were
suspended in RPMI-10% FCS and incubated with viruses at the
concentrations described in the experiments for 6 to 8 h at 37°C
in a 5% CO2 incubator. Ninety-six-well microtiter plates
with a nitrocellulose membrane bottom (Millititer HA plates, Millipore,
Bedford, MA) were coated with ammonium sulfate-precipitated bovine
anti-human IFN- antiserum and fixed with 0.05% glutaraldehyde,
and free aldehyde groups were blocked with 3% glycine in PBS.
Stimulated or mock-stimulated PBMC were washed and added undiluted or
in serial threefold dilutions to the Millititer plates and standard
96-well microtiter plates. The cells were incubated in the plates for
12 to 16 h at 37°C to allow production and capture of the
IFN-. Plates were developed using a murine anti-human IFN-
mAb (293 mAb, provided by Drs. Brita Cederblad and Gunnar Alm, Uppsala,
Sweden) that cross-reacts with multiple IFN- subtypes. This was
followed by incubation with goat antiserum to murine IgG conjugated to
horseradish peroxidase (The Jackson Laboratory, Bar Harbor, ME) and
horseradish peroxidase substrate 3,3'-diaminobenzidine
tetrahydrochloride (Sigma). The resulting reddish brown spots were
enumerated using a dissecting microscope, and the frequencies of IPC
were calculated and expressed as IPC per 104 PBMC.
IFN bioassays
IFN bioassays were performed on supernatants harvested from
standard microtiter plates using a cytopathic effect reduction assay
with GM-0459A cells and VSV as a challenge virus as previously
described (28). IFN- reference standard (National Institute of
Allergy and Infectious Disease standard G-023-901-527) was used at 100
IU/ml.
Isolation of peripheral blood DC by magnetic-activated cell sorting (MACS)
DC were isolated using a blood DC isolation kit (Miltenyi Biotec, Auburn, CA). Briefly, PBMC were resuspended in PBS with 0.5% BSA and 2 mM EDTA (Sigma) and were then labeled with haptenized murine Abs to CD3, CD11b, and CD16 followed by anti-hapten Ab attached to diamagnetic beads. The fraction of cells labeled with magnetic beads (monocytes, NK cells, and T cells) was separated from the nonmagnetic fraction (B cells and DC) using a magnetic separation column. The combined B cell and DC-enriched fraction was then further separated into a B cell and a DC-enriched fraction by labeling with anti-CD4 conjugated to diamagnetic beads. The CD4+ DC were then positively selected on a miniMACS RS+ magnetic separation column, and the nonmagnetic B cells were collected in the flowthrough. DC purity was assessed by flow cytometry, with DC defined as HLA-DR+ cells lacking expression of CD3, CD14, CD16, and CD19. Isolated DC were then used for experiments as described in Results.
RT-PCR quantitation of MR expression
PBMC were sorted into monocyte, NK cell, and T cell-enriched fractions; B cell- and DC-enriched fractions; B cell-enriched fractions; and DC-enriched fractions using the Miltenyi Biotec Blood DC Isolation Kit as described above. Total RNA was then extracted from a fixed number of cells for each population (1 x 105 to 2 x 105) using 0.8 ml of RNAzol (Tel-Test, Friendswood, TX) and 10 µl of microcarrier-GT (Tel-Test). The RNA pellets were dried and resuspended in exactly 10 µl of diethylpyrocarbonate-treated water. cDNA was generated from 2 µl of extracted RNA using Moloney murine leukemia virus reverse transcriptase and the specific downstream antisense primer. RT-PCR was performed according to the manufacturer’s instructions provided with the Perkin-Elmer/Cetus GeneAmp RNA PCR kit (Perkin-Elmer/Cetus, Norwalk, CT). The sequences of the MR-specific primers used were: sense, 5'-CCCTTCCTTGACTAATCC-3'; and antisense, 5'-ACCTCACCCTCC-ACTTATC-3'. This primer set was generated using the GCG software package (Genetics Computer Group, Madison, WI), and the primers were synthesized by the New Jersey Medical School Molecular Resource Facility (Newark, NJ). The sequences of the ß-actin-specific primers used were: sense, 5'-GTGGGGCGCCCCAGGCACCA-3'; and antisense, 5'-GTCCTTAATGTCACGCACGATTTC-3'. This primer set was synthesized by Operon Technologies (Alameda, CA). The PCR reactions for the MR and ß-actin were performed in an 50-µl total reaction volume using the Applied Biosystems 9600 thermocycler (Applied Biosystems, Foster City, CA) and AmpliTaq Gold. cDNA was first kept at 94°C for 10 min to activateAmpliTaq Gold, and amplification was conducted to 40 cycles of 1 min at 94°C, 1 min at 53°C, and 1 min at 72°C per cycle. Forty microliters of each RT-PCR reaction product was then separated on a 2% agarose gel in TBE buffer, and the products (272 kDa for the MR, 520 kDa for ß-actin) were visualized using ethidium bromide staining and a fluorimager (Molecular Devices, Sunnyvale, CA). The PCR product generated with the MR primers was sequenced using an ABI 373 Automated Sequencer (Applied Biosystems) and was found to be 99% homologous with the human MR (data not shown).
Preparation of biotinylated mAb to IFN-
Monoclonal mouse anti-human IFN- (293, a gift from Dr. G.
Alm and B. Cederblad, Uppsala, Sweden) was purified from ascites fluid
using Ultralink-immobilized protein A (Pierce, Rockford, IL) according
to the manufacturer’s instructions. Biotinylation of 293 was performed
using a previously described method (31). Briefly, a 1 mg/ml solution
of 293 in sodium borate buffer (0.1 M, pH 8.8) was prepared by
performing six buffer exchanges (10-fold dilution for each) using a
Centricon 50 spin concentrator (Amicon, Beverly, MA). The Ab was then
reacted with 250 µg of N-hydroxysuccinimide biotin ester
(Sigma) dissolved in DMSO (10 mg/ml stock) for 4 h at room
temperature. Twenty microliters of 1 M NH4Cl was added, and
the Ab was incubated for another 10 min at room temperature. The free
biotin ester was then removed by performing another six buffer
exchanges (10-fold dilution for each) using PBS and a Centricon 50
concentrator. Ab was then stored at 4°C with 0.1% sodium azide until
use.
Preparation of PBMC for intracellular detection of IFN- by flow
cytometry
PBMC were prepared for intracellular detection of IFN- by
flow cytometry using a modification of methods previously described by
Svensson et al. (10). Briefly, PBMC at 2 x 106
cells/ml were stimulated with HSV at 1 x 106 pfu/ml
or mock stimulated for 5 h at 37°C in 5% CO2. The
cells were then washed with PBS and labeled with Ab to the desired
surface determinants or the appropriate isotype control at matched
concentrations for 30 min at 4°C in 50 µl of PBS with 2%
heat-inactivated human serum. The cells were again washed, and then
fixed overnight at 4°C in 1% paraformaldehyde in PBS. Following
fixation, the cells were washed twice in 0.1% BSA in PBS and then
permeabilized for 1 h at room temperature in 0.1% BSA and 0.1%
Tween-20 in PBS. The cells were pelleted and resuspended in the
remaining volume (50 µl) of permeabilization buffer. Biotinylated
293 mAb to IFN- was added to a final concentration of 1 µg/ml, and
the cells were incubated for 30 min at room temperature. The cells were
washed twice and resuspended in the remaining volume, and 10 µl of
streptavidin conjugated to Quantum Red (QR; Sigma) was added. The cells
were incubated for another 30 min, then washed once and resuspended in
300 µl of buffer. For all steps following fixation, the buffer used
was 0.1% BSA and 0.1% Tween-20 in PBS. In some experiments,
nonlabeled 293 Ab was used for intracellular staining of IFN-. In
these cases, a secondary PE-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA) was used for analysis.
Analysis of intracellularly labeled PBMC by flow cytometry
PBMC, stimulated and labeled as described above, were analyzed
using the FACScan flow cytometer and CellQuest Analysis software
(Becton Dickinson, San Jose, CA). PBMC were first gated according to
scatter to include lymphocytes and monocytes within a single region
(region 1), and then data were acquired for cell surface fluorescence
(FITC and PE) and intracellular IFN- (QR). Cells labeled with only a
single fluorochrome were used to adjust compensation and eliminate
overlap among the fluorescence measured by the different channels. IPC
were then defined according to the QR fluorescence using
mock-stimulated cells as a control. IFN--positive cells were then
gated within a second region (region 2), and the cells within regions 1
and 2 were analyzed for surface fluorescence. The percentages of cells
expressing the different surface Ags were then determined by comparison
with the appropriate isotype control using the histogram subtraction
method provided with the Becton Dickinson CellQuest software.
Statistical analysis
Experiments involving monosaccharides and mannan were performed using a randomized block design. The data were analyzed using a two-way ANOVA to account for subject to subject variation, and Student’s t test was used for post-hoc comparisons with adjustment of the p value by a Bonferonni correction. Statistical tests were performed using the JMP statistical software package (SAS Institute, Cary, NC). Curve fitting using the spline method was performed using the Deltagraph 4.0 graphing package (DeltaPoint, Monterey, CA). Statistical analysis of flow cytometric data was conducted by Kolmogorov-Smirnov statistics using Becton Dickinson CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in PBMC
We assessed the abilities of six different monosaccharides to
reduce the frequency of HSV-induced IPC in a dose-dependent manner
(Fig. 1
A). In this
representative experiment, most sugars had inhibitory effects at high
concentrations (> 50 mM). However, Fuc had the greatest inhibitory
effect (IC50 = 25 mM), followed by Gal-N-Ac and Glc-N-Ac
(IC50 
37 mM). Since the specificity of
monosaccharide/lectin interaction is reduced at high concentrations, we
used the sugars in subsequent experiments at a 50-mM concentration.
This concentration was within the linear range of inhibition for all
sugars and corresponded to the approximate IC50 of the Glc
and Gal. As shown in Figure 1B, Fuc was the most inhibitory
monosaccharide reducing the frequency of HSV responsive IPC by 70%,
followed by Gal-N-Ac (60%), Glc-N-Ac (49%), and mannose (31%). The
monosaccharides also decreased the amount of total IFN produced in
response to HSV in a pattern reflecting the frequency changes; the
inhibition was reversible, and cell viability after the 6-h induction
period was >99% by trypan blue exclusion (data not shown). In
contrast to HSV, only Fuc inhibited the induction of IFN by Sendai
virus, yielding a fivefold reduction (Fig. 1C). Since no
receptor with specificity for both Glc-N-Ac/Man and Gal-N-Ac has been
identified, these results suggest either a novel receptor with both
mannose and galactose specificities, or that more than one receptor
with different specificities are involved in the stimulation of IFN-
synthesis by HSV and HIV. Furthermore, the sensitivity of Sendai virus
to only Fuc suggests that Sendai virus stimulates IFN synthesis via a
unique receptor or mechanism.
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production by mannan
Since the results of the monosaccharide inhibition studies
supported a role for lectin receptors in the induction of IFN synthesis
by enveloped viruses, the effect of the yeast cell wall polysaccharide,
mannan, on HSV-induced IFN production in PBMC was examined. Mannans
from a variety of sources are reported to be good ligands for mannose
binding proteins such as the human MR and serum mannose binding protein
(21, 32). In data from five donors (Fig. 2A), mannan achieved a mean
inhibition of approximately 35% at 1 mg/ml; however, this inhibition
was not quite statistically significant (p =
0.0578), most likely due to the large variability in the data. This
donor variability is further exhibited by the results of two donors
shown in Figure 2B in which mannan reduced the IPC frequency
by 40% in one donor and by >90% in another. The lack of complete
inhibition by mannan in most cases further supports that two or more
lectin receptors with different monosaccharide specificities are
involved in the induction process.
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production in PBMC by an
antiserum to the human MR
To further delineate the receptors involved in recognition and
induction of IFN- expression by different enveloped viruses, the
ability of an antiserum specific for the human MR (-MR) to block
virus-induced IFN production was assessed. Figure 3A shows the dose-dependent
effects of anti-MR on the frequency of HSV-responsive IPC. The
antiserum significantly reduced the IPC frequency from the 1/50 to
1/200 dilution in a dose-dependent manner (60–80%) as well as the
total IFN produced at the 1/50 dilution (Fig. 3B),
supporting a role for the MR in the stimulation of IFN- synthesis.
Since HIV and VSV display sensitivities to monosaccharide inhibition
that are similar to that of HSV but that differ from that of Sendai
virus, we examined the blocking effects of -MR on HIV, VSV, and
Sendai virus IFN induction. Results from representative experiments
shown in Figure 4 demonstrate that IFN
induction by HIV and VSV is blocked by anti-MR similar to HSV,
whereas Sendai virus-induced IFN production was unaffected by -MR.
These data further substantiate a common role for the MR in IFN-
induction by different enveloped viruses in PBMC. It also supports
prior studies indicating a separate IFN induction mechanism for Sendai
virus that may correlate with the production of IFN- predominantly
by a CD14+ monocyte that lacks expression of the mannose
and Gal/Gal-N-Ac receptors.
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Although a prior study by Sallusto et al. demonstrated MR expression on cultured DC derived from CD14+ monocytes (26), this receptor has not been reported on DC freshly isolated from blood. RT-PCR and flow cytometry were therefore used to investigate whether freshly isolated peripheral blood DC also express the MR. We have used the Miltenyi dendritic cell isolation kit for these studies. This kit uses negative depletion of T cells, NK cells, and monocytes followed by positive selection of CD4-expressing cells. Typical yields for this procedure are 5 x 105 enriched DC from 108 cells. DC are identified as cells that are MHC class II (HLA-DR) positive but negative for lineage-specific markers of T cells, B cells, NK cells, and monocytes. As expected, the enriched DC are immature, in that they do not express costimulatory molecules or CD83, but up-regulation of both CD80 and CD83 are seen after overnight culture at 37°C (A. Izaguirre and P. Fitzgerald-Bocarsly, in preparation). Wright-Giemsa staining of cytospins of the freshly isolated, enriched DC revealed a homogeneous population of cells that are monocytoid in appearance and size and that lack surface dendritic projections or abundant granules (data not shown), a morphology identical with that previously described for peripheral blood DC and IPC (9, 10, 33).
RT-PCR with primers specific for the MR was performed using PBMC and
cells from each of the steps in the DC isolation protocol. A low level
expression of MR mRNA was observed in unfractionated PBMC (Fig. 5A). A similar level of
expression was observed in cells enriched for T cells, NK cells, and
monocytes, but depleted of B cells and DC, and in cells enriched for B
cells and DC (10-fold enriched for DC). The cell population highly
enriched for DC displayed much higher expression compared with the
other cell fractions (Fig. 5A, lane 5),
demonstrating that the MR is primarily expressed on freshly isolated
DC. The low level expression observed in the DC-depleted fraction
(i.e., T cell-, NK cell-, and monocyte-enriched fraction) may signify
contamination of this fraction by DC, since the T cell-, NK cell-, and
monocyte-enriched fraction is often only partially depleted of IPC
responding to HSV (data not shown).
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D) at levels similar to those
seen with cultured macrophages (27).
While DC express the MR, only a fraction of these DC may be IPC. We
therefore assessed MR expression on IPC using intracellular flow
cytometry for IFN- combined with surface analysis for MR expression
or the binding of an appropriate isotypic control (10). For the
intracellular flow studies we used intact PBMC that were stimulated
with HSV for 5 h at 37°C and then analyzed the phenotype of the
cells positive for intracellular IFN- as has been previously
described by Svensson et al. (10). We chose to use PBMC for these
studies to get an accurate phenotypic profile of the IPC. Moreover,
work by Cederblad et al. has suggested that for optimal production of
IFN-, the HSV-responsive IPC require cytokines or growth factors
that are produced by other cells (34), a finding we have confirmed in
our laboratory. Figure 6 demonstrates
that IPC can be detected by flow cytometry, and they have high forward
light scatter, but low side scatter as previously reported by Svensson
et al. using this technique (10), a scatter profile that is
characteristic of DC. Typically, 0.1 to 0.25% of the PBMC were found
to express intracellular IFN- following stimulation with HSV-1 (for
Fig. 6, 614 cells were positive for IFN- from a total of 350,000
analyzed cells (0.18%)), numbers consistent with those obtained by
ELISPOT determination for HSV-responsive IPC (10, 30). The IFN labeling
was blocked by an excess of exogenous rIFN-2 (Fig. 6, E
and F), demonstrating that this technique is specific for
IFN-. In addition, these cells express HLA-DR and CD4, but have low
to absent expression of CD8 and CD33 (Fig. 7), similar to the pattern previously
described on DC (35) and HSV-stimulated IPC (10). Examination of the MR
expression of the IPC (Fig. 8)
demonstrated that approximately 50% of IPC express the MR
(p < 0.001 compared with isotype control), but
the MR is absent from monocytes and lymphocytes (Fig. 8, D
and E, respectively) as previously reported (21). These data
therefore provide the first evidence that, similar to culture-derived
DC (26), peripheral blood DC and IFN--producing DC express the MR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, we have begun to explore the role of lectin
receptors in viral stimulation of IFN- production by NIPC. We
observed that the frequency of IPC responding to HSV could be decreased
to various degrees by monosaccharides known to block mannose and
galactose binding receptors. Large quantities of sugar were required to
achieve appreciable inhibition, but these quantities were most likely
necessary due to the low affinity binding (Kd =
10-3 M) of monosaccharides for the carbohydrate
recognition domains of lectins compared with the high functional
avidity that occurs with binding to polysaccharide ligands (40). In
addition, these concentrations were similar to those used to study
lectin receptors on NK cells (41, 42). No decrease in cell viability
occurred, and the monosaccharide inhibition was reversible, arguing
against a direct cytotoxic effect of these high monosaccharide
concentrations.
Our results with the polysaccharide, S. cerevisiae mannan,
demonstrate that this substance can also block the induction of IFN-
by HSV. However, the inhibition observed with mannan was highly
variable, with complete inhibition rarely observed. These results as
well as the inhibition observed with monosaccharides that block both
mannose and galactose binding lectins support a DC system with
heterogeneity in the expression of two receptors with differing
specificities. Nevertheless, the possibility that a receptor exists on
DC that binds ligands with both specificities cannot be excluded.
The studies with monosaccharides and mannan lead us to hypothesize that
viruses may induce IFN through interaction with the MR. We evaluated
this possibility and demonstrated that the MR is expressed on a portion
of freshly isolated DC, and approximately half of the cells among PBMC
that stained positively for IFN- (following stimulation with HSV-1)
expressed the MR, a result that was highly significant. Furthermore,
polyclonal antiserum to the MR was found to significantly decrease the
frequency of IPC responding to HSV-1 as well as the amount of IFN-
generated during the viral induction. In future studies, it will be
interesting to isolate and compare the MR-expressing and nonexpressing
IPC.
Since VSV and HIV stimulate the same population of IPC as HSV (4), we
hypothesized that a common receptor may also be involved in induction
of IFN- by these viruses. We evaluated the ability of the
anti-MR antiserum to interfere with IFN induction by VSV, HIV, and
Sendai virus. As hypothesized, the antiserum decreased the frequency of
IPC when induced with VSV and HIV. Although CD4 and chemokine receptors
are known to be the main receptors for HIV, the binding of gp120 to a
mannose binding lectin has previously been reported: HIV was shown to
bind in a CD4-independent manner to a cell-associated mannose binding
lectin in human placenta (43). To date, we have only used HIV-infected
H9 cells in our studies of blocking of IFN- production by
anti-MR Abs. Steinman’s group has reported the interaction of
HIV-1 with dendritic cells occurring via multiple chemokine coreceptors
(44). It will be interesting to determine whether IFN induction by both
T cell- and monocyte-tropic HIV strains are equally blocked by the
anti-MR Ab and whether chemokine receptors are also involved in
this process.
Interestingly, the anti-MR antiserum failed to inhibit IFN-
induction by Sendai virus. This finding is consistent with previously
reported differences between IPC responding to Sendai virus and most
other enveloped viruses, in that Sendai virus induces a high frequency
IPC response that is predominantly monocytic compared with the low
frequency DC response observed with most viruses (4, 5). This
correlates well with the results of the MR expression studies: DC
express the MR, but monocytes do not. Recently, we have also observed
that two compounds, chloroquine and bafilomycin A1, inhibit the
stimulation of IFN- production by viruses that stimulate DC, but
they do not affect IFN- induction by Sendai virus (our unpublished
data). Both drugs raise endosomal pH by independent mechanisms (45, 46), and their inhibitory effects may relate to interruption of normal
lysosomal functioning or MR recycling through this compartment of the
cell. Since Sendai virus infection can occur by extracellular envelope
fusion and its induction of IFN is not inhibited by blocking the MR,
these results may reflect the unique ability of this virus to stimulate
monocytes.
The MR plays a role in the induction of IFN- production by DC;
however, some evidence suggests that additional mechanisms act in
combination with or independently of the MR. Most obvious is the
inhibition observed with Gal-N-Ac. This sugar poorly blocks the MR, but
is an excellent ligand for lectins with Gal/Gal-N-Ac specificity (47).
Recently, a human receptor that binds Gal and Gal-N-Ac has been cloned
in humans that is expressed on macrophages (23), and it will be
interesting to examine its expression on DC.
The precise function of the MR in the induction of IFN- remains to
be elucidated. One plausible scheme involves binding of the virus using
the MR or other receptors. Following binding, the virus is
internalized, resulting in either productive or nonproductive
infection. The stimulus for IFN- synthesis may then be derived from
the viral nucleic acid. This model is supported by evidence that IFN
induction correlates with virulence in some systems, and both dsRNA and
DNA containing certain palindromic sequences can induce IFN (48). Some
evidence, however, suggests that viruses may be capable of inducing IFN
by simply binding to a surface receptor that can transduce a signal for
IFN- synthesis independent of infection (15, 49). Again, the MR may
be a candidate receptor given its ability to transduce a mitogenic
signal in alveolar smooth muscle cells in response to the appropriate
ligand (50).
Finally, lectin receptors may serve as a universal, nonspecific means
by which DC recognize many different viral and nonviral pathogens.
Following binding and internalization, these pathogens would be
shuttled to the MHC class II-rich endosomal compartment of DC for Ag
processing and ultimately presentation (26), and some of these
pathogens may then induce the production of IFN- and other mediators
by the DC. The IFN may serve as a local antiviral substance protecting
neighboring cells and activating NK cells and macrophages (38, 51).
However, the IFN- would also serve as an important modulator of T
cell activation and maintenance of T cell memory during the
presentation process. The interaction of virus with lectin receptors on
DC may therefore serve as a critical link between the innate and
adaptive immune responses to the myriad of pathogens encountered.
|
Acknowledgments |
|---|
, and Wellcome-Glaxo for providing polyclonal
antiserum to IFN-. We also acknowledge Drs. Sheela Amrute and Robert
Donnelly of the New Jersey Medical School for the sequencing of the MR
RT-PCR product, and Dana Stein of New Jersey Medical School for
assistance with flow cytometry. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Patricia Fitzgerald-Bocarsly, Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103. E-mail address:
3 Abbreviations used in this paper: HSV, herpes simplex virus; NIPC, natural interferon--producing cells; VSV, vesicular stomatitis virus; DC, dendritic cell; IPC, interferon-producing cell(s); MR, mannose receptor; Glc, D-glucose; Gal, D-galactose; Man, D-mannose; Glc-N-Ac, N-acetylglucosamine; Gal-N-Ac, N-acetylgalactosamine; Fuc, fucose; NGS, normal goat serum; PE, phycoerythrin; ELISPOT, enzyme-linked immunospot; MACS, magnetic-activated cell sorting; QR, Quantum Red; pfu, plaque-forming unit.
Received for publication January 15, 1998. Accepted for publication April 30, 1998.
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) or
the presence (•) of different dilutions of anti-MR antiserum or
NGS, and the frequency of IPC was determined by ELISPOT assay. The data
displayed for each virus are representative of at least three donors
and are expressed as a percentage of the control IPC frequency for PBMC
stimulated in the absence of antiserum.
