Download Maturation and function of human dendritic cells are inhibited by orf

Journal of General Virology (2006), 87, 3177–3181
Short
Communication
DOI 10.1099/vir.0.82238-0
Maturation and function of human dendritic cells
are inhibited by orf virus-encoded interleukin-10
Anna Chan, Margaret Baird, Andrew A. Mercer and Stephen B. Fleming
Correspondence
Stephen Fleming
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin,
New Zealand
stephen.fleming@stonebow.
otago.ac.nz
Received 25 May 2006
Accepted 11 July 2006
Orf virus (ORFV) is a parapoxvirus that infects sheep, goats and man. In humans, the virus induces
acute, pustular skin lesions that can develop into a progressive disease. Humans are susceptible to
reinfection with ORFV and rare cases of persistent infection have been reported. ORFV
encodes several immunomodulators, including a homologue of interleukin-10 (ORFV IL-10), that
may explain these phenomena. The immunosuppressive effects of ORFV IL-10 on immature
human dendritic cells (DCs) cultured from blood-derived monocytes (MoDCs) were investigated.
MoDCs exposed simultaneously to lipopolysaccharide and ORFV IL-10 showed enhanced
ovalbumin–FITC uptake and reduced IL-12 expression, indicating inhibition of maturation.
Moreover, ORFV IL-10 inhibited the upregulation of DC cell-surface activation and maturation
markers MHC II, CD80, CD83 and CD86 and inhibited the capacity of MoDCs to activate CD4+
T cells in an oxidative mitogenesis assay. These findings suggest that ORFV IL-10 may influence the
development of acquired immunity in humans by impairing DC function.
Orf virus (ORFV) is a parapoxvirus that infects sheep and
goats, but is transmissible to man. In humans, the virus
induces acute, pustular skin lesions that can develop into a
progressive disease in the form of multiple lesions (Sanchez
et al., 1985). In immune-impaired individuals, large, highly
vascularized, tumour-like lesions of the skin have been
reported (Savage & Black, 1972; Tan et al., 1991). ORFV
infections can cause complications such as erythema
multiforme reactions (Agger & Webster, 1983; Blakemore
et al., 1948; Fastier, 1957; Mourtada et al., 2000) and severe
forms of erythema multiforme have been reported (Erickson
et al., 1975). In addition, ORFV can readily reinfect its hosts,
in spite of apparently normal host antivirus immune and
inflammatory responses (Haig & McInnes, 2002; Robinson
& Lyttle, 1992). This phenomenon has raised questions
about the mechanisms underlying this apparent escape from
immunity.
ORFV encodes a number of immunomodulators, which
include a homologue of interleukin-10 (ORFV IL-10)
(Fleming et al., 1997; Mercer et al., 2006). Mammalian IL10 is a multifunctional cytokine that is a potent suppressor
of inflammation and cross-regulates a type 1 cell-mediated
response (Moore et al., 2001). ORFV IL-10 is 80 % identical
at the amino acid level to ovine IL-10, suggesting that the
gene has been captured from sheep and has adapted to this
A figure showing characterization of MoDCs and a table showing the
effect of ORFV IL-10 on IL-12 production from MoDCs are available as
supplementary material in JGV Online.
0008-2238 G 2006 SGM
species. In comparison, ORFV IL-10 is 67 % identical at the
amino acid level to human IL-10 (hIL-10), with differences
in amino acids that are likely to be critical for interaction of
ORFV IL-10 with the human IL-10 receptor (Fickenscher
et al., 2002; Josephson et al., 2001). We have previously
characterized ORFV IL-10 activities in ovine cells and
murine cells and shown that it is functionally similar to
mammalian IL-10 (Fleming et al., 1997, 2000; Haig et al.,
2002; Imlach et al., 2002; Lateef et al., 2003).
The acquired immune response is initiated by dendritic cells
(DCs) that also regulate the quality and magnitude of the
immune response. DCs are disrupted at multiple stages by
IL-10 (De Smedt et al., 1997; Faulkner et al., 2000; Moore
et al., 2001). IL-10 inhibits the activation and maturation
of DCs, manifested as the inability to upregulate major
histocompatibility complex (MHC) class II and costimulatory molecules such as CD80, CD83 and CD86, reduced IL12 production and reduced ability to activate and present
antigen to T cells. Here, we report the immunosuppressive
effects of ORFV IL-10 on human monocyte-derived DCs
(MoDCs) in vitro.
Human immature MoDCs were generated from peripheral
blood mononuclear cells (PBMCs) from healthy donors
(approved by the Otago University Ethics Committee).
PBMCs were isolated as described previously (Copland
et al., 2003). The adherent monocytes were cultured in
DC medium containing 25 ng ml21 each of recombinant
human granulocyte–macrophage colony-stimulating factor
(GM-CSF) and IL-4 (R&D Systems). On day 2, a further
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A. Chan and others
2 ml DC medium was added to cells. After 5 days
culture, non-adherent immature MoDCs were seeded at
66105 cells ml21 before use.
MoDCs that were generated in vitro were analysed by using
flow cytometry as described previously (Copland et al.,
2003). MoDCs exhibited forward (FSC-H) and side (SSCH) scatter characteristics of DCs and the majority of the cells
lay in the R1 gate (see Supplementary Fig. S1, available in
JGV Online). MoDCs in the R1 gate were further analysed
for expression of CD11c, a specific marker for MoDCs
(Freudenthal & Steinman, 1990; Shortman & Liu, 2002),
with allophycocyanin-conjugated anti-CD11c (S-HCL-3;
IgG2b; BD Biosciences). MoDCs were gated on this marker
for all subsequent analyses. Over a number of donors (more
than eight), the proportion of cells in culture that were
CD11c+ (MoDCs) was between 46 and 89 % and >95 % of
cells in the R1 gate were CD11c+. The MoDCs generated
had a typical immature phenotype, as shown by the low
percentage of cells expressing the cell-surface activation
markers CD80, CD83 and CD86, and almost all MoDCs
expressed MHC II. The mouse anti-human mAbs used were:
phycoerythrin-conjugated anti-HLA-DR (MHC II) (clone
L243; isotype IgG2a; BD Biosciences), anti-CD80 (L307.4;
IgG1k Pharmingen), anti-CD83 (HB15e; IgG1; Caltag) and
anti-CD86 (BU63; IgG1; Serotech).
As DCs mature, they lose their ability to acquire antigen. We
examined whether ORFV IL-10 was able to inhibit MoDC
maturation induced by lipopolysaccharide (LPS) by quantifying the uptake of the antigen fluorescein isothiocyanatelabelled ovalbumin (FITC–OVA) (Copland et al., 2003).
Immature MoDCs were treated with hIL-10 (R&D Systems)
or affinity-purified FLAG-tagged ORFV IL-10 (Imlach et al.,
2002) and with or without LPS (Escherichia coli O26 : B6;
Sigma) for 24 h. The optimal amount of ORFV IL-10 used in
these assays (50 ng ml21) was determined in preliminary
experiments (data not shown) and in addition by examining
its ability to inhibit cytokine synthesis in LPS-activated THP1 cells (human monocytes) (L. M. Wise, C. A. McCaughan &
S. B. Fleming, unpublished data). MoDCs were then
incubated with 40 mg FITC–OVA ml21 (Lateef et al.,
2003), labelled for CD11c and analysed by flow cytometry.
CD11c+ MoDCs treated with ORFV IL-10 in the presence of
LPS retained their ability to take up antigen and the levels
determined were similar to those for immature MoDCs not
exposed to LPS (Fig. 1). Statistical analysis (single-factor
analysis of variance and Tukey’s test) using the combined
data for the three donors showed a statistically significant
difference for antigen uptake between cells treated with
ORFV IL-10/LPS or hIL-10/LPS and cells exposed to LPS
only (P<0?05). There was no significant difference between
the effects of ORFV IL-10/LPS and hIL-10/LPS (P>0?05).
Supernatants collected from the above cells, prior to flowcytometry analysis, were analysed for IL-12p70 by ELISA
(Pharmingen). IL-12 was detected (305–631 pg ml21)
where cells were activated with LPS and then exposed to
FITC–OVA, but not where ORFV IL-10 or hIL-10 was added
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Fig. 1. Effect of ORFV IL-10 on antigen uptake by MoDCs.
MoDCs were treated simultaneously with hIL-10 or ORFV IL10 (50 ng ml”1) in the presence or absence of LPS
(10 ng ml”1) for 24 h. MoDCs were then incubated with FITC–
OVA for 2 h and analysed by flow cytometry. MoDCs that were
not exposed to hIL-10, ORFV IL-10, LPS or FITC–OVA are
shown as MoDC. MoDCs that were only exposed to FITC–
OVA are shown as FITC control. Antigen uptake is shown as
median fluorescence intensity (MFI). Filled bars, donor R;
shaded bars, donor V; empty bars, donor Y.
to cells in addition to LPS/FITC–OVA (see Supplementary
Table S1, available in JGV Online). As we were detecting IL12 from a heterogeneous population of cells, we could not be
certain whether IL-12 was produced by CD11c+ cells or from
other cell types. Nevertheless, it was apparent that both IL-10s
were potent inhibitors of IL-12 production.
We then examined whether ORFV IL-10 inhibited the
maturation of human DCs in the presence of an activation
signal by examining changes in cell-surface phenotypic
markers that characterize mature DCs. MoDCs were
cultured for 24 h with or without ORFV IL-10 in the
presence of LPS, double-stained with antibodies against
CD11c and MHC II, CD80, CD83 and CD86 and analysed
by flow cytometry. Ten thousand cells were collected from
each sample and analysed by using CellQuest Pro software
(BD Biosciences).
Addition of ORFV IL-10 during LPS activation appeared to
inhibit the upregulation of MHC II and was most marked in
cells from donor W, which showed a 4?8-fold reduction in
MHC II expression in cells treated with LPS and ORFV IL10 compared with cells treated with LPS only (Fig. 2a).
MoDCs derived from donor R showed a 2?2-fold reduction
in MHC II expression, whilst MoDCs derived from donor T
showed readily distinguishable, but lower, inhibition. There
was little difference in the percentage of cells that stained
CD11c+ MHC II+ for all treatments (data not shown).
There was no significant difference (P>0?05) for the
combined data from the three donors for CD11c+ MHC II
expression for cells treated with LPS compared with cells
treated with LPS/ORFV IL-10, which is due to the smaller
differences seen in donor T.
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Journal of General Virology 87
ORFV IL-10 inhibits DC maturation and function
over 90 % of cells treated with LPS expressed this marker.
Where cells were treated simultaneously with LPS and
ORFV IL-10, the proportion of cells that stained positive for
CD80 varied from 37 % (donor W) to 88 % (donor R). The
proportion of untreated immature MoDCs expressing the
CD83 activation marker was <5 %, but increased to
between 32 and 48 % on stimulation with LPS alone.
With cells treated simultaneously with IL-10 and LPS, the
proportion of cells expressing the activation marker CD83
was <17 %. The proportion of untreated immature cells
expressing CD86 was also low (<8 %), but increased to
58–81 % when cells were activated with LPS alone. In
contrast, with cells treated simultaneously with LPS and
ORFV IL-10, the proportion of cells expressing CD86 was
between 5 and 22 %. Statistical analysis for the combined
data of the three donors showed that there was no significant
difference in CD80 expression for cells treated with LPS
compared with cells treated with ORFV IL-10/LPS or hIL10/LPS (P>0?05), whereas there was a significant difference
for CD83 and CD86 expression (P<0?05). Overall, ORFV
IL-10 and hIL-10 had similar inhibitory effects on MoDC
phenotypic-marker expression.
We then investigated the effects of ORFV IL-10 on the ability
of MoDCs to activate T cells in a functional oxidative
mitogenesis assay. This assay measures the capacity of
MoDCs to induce CD4+ T-cell proliferation (Gunzer et al.,
2000; Thoeni et al., 2005). CD4+ T cells were purified from
PBMCs by using BD IMag anti-human CD4 particles-DM
(clone L200) according to the manufacturer’s instructions
(BD Biosciences) (final suspension, >84 % CD4+). CD4+
T cells (16107 ml21) were treated with 0?25 mg sodium
periodate ml21 (Sigma), washed twice and seeded at
2?56105 per well. Pre-treated MoDCs were harvested,
washed twice and 2?56104 pre-treated MoDCs were then
added to CD4+ T cells and incubated for 24 h. The amount
of [3H]thymidine incorporated was determined 20 h after
addition to the culture by using a 1450 Microbeta liquid
scintillation counter.
Fig. 2. Effect of ORFV IL-10 on the phenotype of LPS-activated
MoDCs. MoDCs were treated simultaneously with hIL-10 or
ORFV IL-10 (50 ng ml”1) in the presence or absence of LPS
(10 ng ml”1) for 24 h and analysed by flow cytometry. (a) Degree
of MHC II expression (MFI). Filled bars, donor R; shaded bars,
donor T; empty bars, donor W. (b) Percentage of CD11c+
MoDCs expressing CD80, CD83 and CD86. MoDC indicates
cells not exposed to hIL-10, ORFV IL-10 or LPS (control). Empty
bars, CD80; shaded bars, CD83; filled bars, CD86.
The inhibitory effects of ORFV IL-10 were also seen in the
numbers of cells expressing CD80, CD83 and CD86
(Fig. 2b). The proportion of untreated MoDCs expressing
CD80 varied from 20 to 46 % over the three donors, whereas
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In preliminary experiments, LPS-treated MoDCs were
titrated against purified CD4+ T cells to obtain the optimal
ratio for the oxidative mitogenesis assay. From the titrations
of MoDCs, a ratio of 1 : 10 (MoDCs : CD4+ T cells) was used
for subsequent assays (data not shown). The ability of ORFV
IL-10 to modulate CD4+ T-cell activation by MoDCs
indirectly was then examined. Our results showed that
whereas LPS-matured MoDCs stimulated significantly
higher proliferation than did untreated MoDCs (P<0?05,
Student’s t-test), those MoDCs pretreated with ORFV IL-10
were unable to do this (P>0?05; Fig. 3). The CD4+ T-cell
proliferation induced by these MoDCs was comparable to
that induced by MoDCs treated with hIL-10 and LPS or
to that induced by untreated MoDCs. Similar results
were obtained by using autologous T cells or allogeneic
T cells. These results suggest that ORFV IL-10-mediated
inhibition of DC maturation impairs their ability to
stimulate T cells.
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A. Chan and others
persistent infection in some normal individuals (Pether et al.,
1986; Rogers et al., 1989) and East Fresian sheep (S. B.
Fleming & A. A. Mercer, unpublished data) suggests that this
is the case. The effects of ORFV IL-10 on DCs could affect
both the innate responses and the acquired immune response
in several ways. It could impair the production of IL-12 by
DCs, which in turn will result in poor production of IFN-c by
NK and NKT cells. It could lead to a delay in the development
of acquired immunity, skew the immune response from an
antiviral Th1 response towards a Th2 response (Chang et al.,
2004) or lead to immunological tolerance (Lutz & Schuler,
2002) or anergy (Raftery et al., 2004).
Fig. 3. Effect of ORFV IL-10 on the ability of MoDCs to activate CD4+ T cells. MoDCs (2?56104) treated with hIL-10 or
ORFV IL-10 (50 ng ml”1), with and without LPS (10 ng ml”1)
for 24 h, were co-cultured with 2?56105 CD4+ T cells for
24 h. Experiments were performed with autologous cells except
where indicated. Assays were carried out in triplicate.
Proliferation was measured by incorporation of [3H]thymidine
during the last 20 h of culture. Results are expressed as mean
c.p.m.±SD. Significant differences were relative to LPS control.
*P<0?05. MoDC indicates cells not exposed to hIL-10, ORFV
IL-10 or LPS (control).
The results of our study showed unequivocally that ORFV
IL-10 has inhibitory effects on the maturation of human
MoDCs, despite some variation in responses that may
reflect genetic differences, assay-to-assay variation or
maturation status of cells at the time of isolation (Morel
et al., 1997). In all assays performed, with the exception of
MHC II expression and CD80 expression, statistically
significant differences were demonstrated between donors.
Surprisingly, differences in the polypeptide sequence
between ORFV IL-10 and hIL-10 did not translate into
functional differences for these cytokines and will be
discussed in detail elsewhere.
DCs represent a strategically important target for immune
evasion by viruses that cause persistent infections, such as
herpesviruses, which produce secreted versions of IL-10. It is
unusual for viruses that cause acute infections to produce
factors that target DCs specifically; however, the fact that
ORFV reinfects sheep and humans and is able to establish
3180
We predict that ORFV IL-10 is suppressing inflammation
early in infection, in conjunction with other anti-inflammatory viral factors, and thus delaying the recruitment of
DCs to the site of infection. Whilst there is no evidence that
Langerhans cells are involved in ORFV infection, other DC
subsets, such as dermal or blood-derived DCs, could play
important roles (Lear et al., 1996; Villadangos & Heath,
2005). Immature DCs that reach the site of infection and
take up antigen could be prevented from maturing by the
presence of ORFV IL-10. The additional consequence of this
effect could be that it also serves to clear inflammatory
stimuli from the site of inflammation (Grütz, 2005). In
addition, there could be other mechanisms acting that
destroy DCs. Cytomegalovirus IL-10 has been shown to
increase apoptosis associated with DC maturation (Chang
et al., 2004; Raftery et al., 2004). We have observed similar
effects when human MoDCs were treated with ORFV IL-10
over an extended period (data not shown). In cases of
reinfection, it is possible that the same mechanisms operate,
as there is no evidence that Th1 memory is impaired in ovine
species, as shown by a strong delayed hypersensitivity
response to ORFV antigen (Buddle & Pulford, 1984).
In conclusion, we have shown, by demonstrating the
inhibitory effects of ORFV IL-10 on human DC maturation
and its indirect effects on T-cell activation, that it has the
potential to impair DCs over many stages of functionality. We
propose that ORFV IL-10, in conjunction with other ORFV
immunomodulators that have activity on human cells and
signalling molecules, causes a delay in the development of
cell-mediated immunity during primary exposure and
reinfection. Furthermore, the expression of immunomodulators such as viral IL-10 may provide an immunological basis
for rare cases of persistent ORFV pathology in humans.
Acknowledgements
We thank Catherine McCaughan, Michelle Wilson and Dr Sarah
Young for their excellent technical help and advice and the Health
Research Council of New Zealand for funding this project.
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