| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Stephen Fleming
stephen.fleming{at}stonebow.otago.ac.nz
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
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.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). 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 IL-10 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 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 IL-12 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 ml–1 each of recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 (R&D Systems). On day 2, a further 2 ml DC medium was added to cells. After 5 days culture, non-adherent immature MoDCs were seeded at 6x105 cells ml–1 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 (SSC-H) 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; IgG1
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 isothiocyanate-labelled 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 ml–1) was determined in preliminary experiments (data not shown) and in addition by examining its ability to inhibit cytokine synthesis in LPS-activated THP-1 cells (human monocytes) (L. M. Wise, C. A. McCaughan & S. B. Fleming, unpublished data). MoDCs were then incubated with 40 µg FITC–OVA ml–1 (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).
|
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 IL-10 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.
|
). The proportion of untreated MoDCs expressing CD80 varied from 20 to 46 % over the three donors, whereas 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 hIL-10/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 (1x107 ml–1) were treated with 0.25 mg sodium periodate ml–1 (Sigma), washed twice and seeded at 2.5x105 per well. Pre-treated MoDCs were harvested, washed twice and 2.5x104 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.
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.
|
). 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 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-
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).
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 |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Blakemore, F., Abdussalam, M. & Goldsmith, W. N. (1948). A case of orf (contagious pustular dermatitis): identification of the virus. Br J Dermatol 60, 404–409.[CrossRef]
Buddle, B. M. & Pulford, H. D. (1984). Effect of passively-acquired antibodies and vaccination on the immune response to contagious ecthyma virus. Vet Microbiol 9, 515–522.[CrossRef][Medline]
Chang, W. L., Baumgarth, N., Yu, D. & Barry, P. A. (2004). Human cytomegalovirus-encoded interleukin-10 homolog inhibits maturation of dendritic cells and alters their functionality. J Virol 78, 8720–8731.
Copland, M. J., Baird, M. A., Rades, T., McKenzie, J. L., Becker, B., Reck, F., Tyler, P. C. & Davies, N. M. (2003). Liposomal delivery of antigen to human dendritic cells. Vaccine 21, 883–890.[CrossRef][Medline]
De Smedt, T., Van Mechelen, M., De Becker, G., Urbain, J., Leo, O. & Moser, M. (1997). Effect of interleukin-10 on dendritic cell maturation and function. Eur J Immunol 27, 1229–1235.[Medline]
Erickson, G. A., Carbrey, E. A. & Gustafson, G. A. (1975). Generalized contagious ecthyma in a sheep rancher: diagnostic considerations. J Am Vet Med Assoc 166, 262–263.[Medline]
Fastier, L. B. (1957). Human infections with the virus of ovine contagious pustular dermatitis (scabby mouth). N Z Med J 56, 121–123.[Medline]
Faulkner, L., Buchan, G. & Baird, M. (2000). Interleukin-10 does not affect phagocytosis of particulate antigen by bone marrow-derived dendritic cells but does impair antigen presentation. Immunology 99, 523–531.[CrossRef][Medline]
Fickenscher, H., Hör, S., Küpers, H., Knappe, A., Wittmann, S. & Sticht, H. (2002). The interleukin-10 family of cytokines. Trends Immunol 23, 89–96.[CrossRef][Medline]
Fleming, S. B., McCaughan, C. A., Andrews, A. E., Nash, A. D. & Mercer, A. A. (1997). A homolog of interleukin-10 is encoded by the poxvirus orf virus. J Virol 71, 4857–4861.[Abstract]
Fleming, S. B., Haig, D. M., Nettleton, P., Reid, H. W., McCaughan, C. A., Wise, L. M. & Mercer, A. A. (2000). Sequence and functional analysis of a homolog of interleukin-10 encoded by the parapoxvirus orf virus. Virus Genes 21, 85–95.[CrossRef][Medline]
Freudenthal, P. S. & Steinman, R. M. (1990). The distinct surface of human blood dendritic cells, as observed after an improved isolation method. Proc Natl Acad Sci U S A 87, 7698–7702.
Grütz, G. (2005). New insights into the molecular mechanism of interleukin-10-mediated immunosupression. J Leukoc Biol 77, 3–15.
Gunzer, M., Schäfer, A., Borgmann, S., Grabbe, S., Zänker, K. S., Bröcker, E.-B., Kämpgen, E. & Friedl, P. (2000). Antigen presentation in extracellular matrix: interactions of T cells with dendritic cells are dynamic, short lived, and sequential. Immunity 13, 323–332.[CrossRef][Medline]
Haig, D. M. & McInnes, C. J. (2002). Immunity and counter-immunity during infection with the parapoxvirus orf virus. Virus Res 88, 3–16.[CrossRef][Medline]
Haig, D. M., Thomson, J., McInnes, C. J. & 7 other authors (2002). A comparison of the anti-inflammatory and immuno-stimulatory activities of orf virus and ovine interleukin-10. Virus Res 90, 303–316.[CrossRef][Medline]
Imlach, W., McCaughan, C. A., Mercer, A. A., Haig, D. & Fleming, S. B. (2002). Orf virus-encoded interleukin-10 stimulates the proliferation of murine mast cells and inhibits cytokine synthesis in murine peritoneal macrophages. J Gen Virol 83, 1049–1058.
Josephson, K., Logsdon, N. J. & Walter, M. R. (2001). Crystal structure of the IL-10/IL-10R1 complex reveals a shared receptor binding site. Immunity 15, 35–46.[CrossRef][Medline]
Lateef, Z., Fleming, S., Halliday, G., Faulkner, L., Mercer, A. & Baird, M. (2003). Orf virus-encoded interleukin-10 inhibits maturation, antigen presentation and migration of murine dendritic cells. J Gen Virol 84, 1101–1109.
Lear, A., Hutchison, G., Reid, H. W., Norval, M. & Haig, D. M. (1996). Phenotypic characterisation of the dendritic cells accumulating in ovine dermis following primary and secondary orf virus infections. Eur J Dermatol 6, 135–140.
Lutz, M. B. & Schuler, G. (2002). Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23, 445–449.[CrossRef][Medline]
Mercer, A. A., Ueda, N., Friederichs, S.-M., Hofmann, K., Fraser, K. M., Bateman, T. & Fleming, S. B. (2006). Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation. Virus Res 116, 146–158.[Medline]
Moore, K. W., de Waal Malefyt, R., Coffman, R. L. & O'Garra, A. (2001). Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19, 683–765.[CrossRef][Medline]
Morel, A. S., Quaratino, S., Douek, D. C. & Londei, M. (1997). Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step. Eur J Immunol 27, 26–34.[Medline]
Mourtada, I., Le Tourneur, M., Chevrant-Breton, J. & Le Gall, F. (2000). Human orf and erythema multiforme. Ann Dermatol Venereol 127, 397–399 (in French).[Medline]
Pether, J. V., Guerrier, C. J., Jones, S. M., Adam, A. E. & Kingsbury, W. N. (1986). Giant orf in a normal individual. Br J Dermatol 115, 497–499.[CrossRef][Medline]
Raftery, M. J., Wieland, D., Gronewald, S., Kraus, A. A., Giese, T. & Schönrich, G. (2004). Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-10. J Immunol 173, 3383–3391.
Robinson, A. J. & Lyttle, D. J. (1992). Parapoxviruses: their biology and potential as recombinant vaccines. In Recombinant Poxviruses, pp. 285–327. Edited by M. M. Binns & G. L. Smith. Boca Raton, FL: CRC Press.
Rogers, M., Bale, P., De Silva, L. M., Glasson, M. J. & Collins, E. (1989). Giant parapox infection in a two year old child. Australas J Dermatol 30, 87–91.[Medline]
Sanchez, R. L., Hebert, A., Lucia, H. & Swedo, J. (1985). Orf. A case report with histologic, electron microscopic, and immunoperoxidase studies. Arch Pathol Lab Med 109, 166–170.[Medline]
Savage, J. & Black, M. M. (1972). ‘Giant’ orf of finger in a patient with a lymphoma. Proc R Soc Med 65, 766–768.[Medline]
Shortman, K. & Liu, Y.-J. (2002). Mouse and human dendritic cell subtypes. Nat Rev Immunol 2, 151–161.[CrossRef][Medline]
Tan, S. T., Blake, G. B. & Chambers, S. (1991). Recurrent orf in an immunocompromised host. Br J Plast Surg 44, 465–467.[CrossRef][Medline]
Thoeni, G., Stoitzner, P., Brandacher, G., Romani, N., Heufler, C., Werner-Felmayer, G. & Werner, E. R. (2005). Tetrahydro-4-aminobiopterin attenuates dendritic cell-induced T cell priming independently from inducible nitric oxide synthase. J Immunol 174, 7584–7591.
Villadangos, J. A. & Heath, W. R. (2005). Life cycle, migration and antigen presenting functions of spleen and lymph node dendritic cells: limitations of the Langerhans cells paradigm. Semin Immunol 17, 262–272.[CrossRef][Medline]
Received 25 May 2006;
accepted 11 July 2006.
This article has been cited by other articles:
|
|
 |
|
  S. B. Fleming, I. E. Anderson, J. Thomson, D. L. Deane, C. J. McInnes, C. A. McCaughan, A. A. Mercer, and D. M. Haig Infection with recombinant orf viruses demonstrates that the viral interleukin-10 is a virulence factor J. Gen. Virol., July 1, 2007; 88(7): 1922 - 1927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wise, C. McCaughan, C. K. Tan, A. A. Mercer, and S. B. Fleming Orf virus interleukin-10 inhibits cytokine synthesis in activated human THP-1 monocytes, but only partially impairs their proliferation J. Gen. Virol., June 1, 2007; 88(6): 1677 - 1682. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
