Institut für Virologie der Universität Würzburg, Versbacher Str.7, 97078 Würzburg, Germany1
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, MT 59840, Hamilton, USA2
Author for correspondence: Ulf Dittmer. Fax +49 931 2013934. e-mail ulf.dittmer{at}mail.uni-wuerzburg.de
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Abstract |
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FV is a complex comprised of a replication-competent helper virus known as Friend murine leukaemia virus (F-MuLV), and a replication-defective but pathogenic virus, spleen focus forming virus (SFFV) (Kabat, 1989 ). Infection of adult mice with FV induces acute viraemia and splenomegaly of various degrees depending on the genetic background of the mouse strain (Chesebro et al., 1990
; Hasenkrug, 1999
). In susceptible strains, disease progresses to lethal erythroleukaemia (Moreau-Gachelin et al., 1988
; Munroe et al., 1990
; Wendling & Tambourin, 1978
). Both virus-specific cellular and humoral immune responses are essential for recovery from primary FV infection (Hasenkrug et al., 1995
; Hasenkrug & Chesebro, 1997
; Robertson et al., 1992
; Super et al., 1998
). Infection of mice with F-MuLV helper virus alone protects mice from subsequent challenge with FV complex because the helper virus replicates poorly in the absence of SFFV, contains important immunological determinants and thus acts as a live attenuated vaccine virus (Dittmer et al., 1998
). Previous experiments showed that protection with this live attenuated virus requires complex immune responses including CD4+ T cells (Th); CD8+ T cells (CTL) and B cells (antibody-producing cells) (Dittmer et al., 1999b
).
In the current study, we analysed the role of IL-5, IL-6 and IL-10 in immunity to FV infection using mice with genetic inactivations in each of these cytokine genes. Experiments using mice with deficiencies in a specific cytokine have proven to be useful models for obtaining information about the regulation of immune cells in response to infection. All mice used for our experiments were on the C57BL/6 (B6) genetic background because of the availability of cytokine genetic inactivations in this mouse strain. B6 mice are genetically resistant to FV-induced erythroleukaemia due to the Fv2 gene, which acts in a non-immunological manner to limit FV-induced polyclonal cell activation and splenomegaly (Hoatlin & Kabat, 1995 ; Persons et al., 1999
). Despite the genetic resistance of normal B6 mice to FV-induced disease, they are not able to completely eradicate Friend virus, and infection causes a low level, life-long, persistent infection. Furthermore, experiments have shown that B6 mice deficient in specific lymphocyte subsets, such as CD4+ or CD8+ T cells, develop late-onset lethal erythroleukaemia (Hasenkrug, 1999
). Thus, part of the resistance of B6 mice to FV-induced erythroleukaemia is mediated by specific lymphocytes, and this resistance likely involves the production of cytokines. We previously showed that it was possible to prevent the establishment of persistent FV infections as well as prevent acute disease by vaccinating mice with a live attenuated Friend helper virus (Dittmer et al., 1999a
). Such vaccine-induced protection from persistent infections was shown to be associated with complete clearance of infectious centres from the spleen by 2 weeks post-challenge. Therefore, in the current studies we analysed the role of IL-5, IL-6 and IL-10 in vaccine-induced clearance of spleen virus by 2 weeks post challenge, as well as the role of these cytokines in the resolution of acute infection.
To determine whether the B-cell stimulating cytokines IL-5 and IL-6 were important in primary FV-specific immune responses, virus loads in the plasma and spleens (for methods see Hasenkrug et al., 1998a , b
) of FV-infected B6 wild-type mice were compared with those of cytokine-deficient mice. At 1 week post-infection, all wild-type B6 mice were viraemic, with levels between 103 to 104 focus forming units (f.f.u.) per ml of plasma (mean=7762 f.f.u., SD=669 f.f.u., n=8). Both IL-5- (mean=10232 f.f.u., SD=1279 f.f.u., n=13) and IL-6-deficient mice (mean=2692 f.f.u., SD=277 f.f.u., n=14) had levels of viraemia in the same range as wild-type mice. In contrast, at 2 weeks post-FV infection, the spleen virus loads were significantly higher in IL-6-/- mice than in B6 controls (Fig. 1A
). Thus, IL-6 appeared to have an important function in restricting early FV replication. In contrast, the comparison of IL-5-/- with wild-type B6 mice did not reveal any significant differences in cell-associated virus loads (Fig. 1A
). In addition, we also analysed the influence of the immunomodulating cytokine IL-10 on virus loads of FV-infected mice. Deficiency in IL-10 did not have a statistically significant influence on viraemia levels at 1 week post-infection (mean=12022 f.f.u., SD=1123 f.f.u., n=16). However, at 2 weeks post-infection the cell-associated virus loads in the spleens were about 10 times higher in IL-10-/- mice than in wild-type animals (Fig. 1A
). Thus, IL-10 deficiency led to impaired virus control rather then enhanced antiviral immune responses.
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In previous experiments we used N-tropic F-MuLV helper virus as a vaccine virus to prevent acute viraemia (Dittmer et al., 1998 ) and persistent FV infection (Dittmer et al., 1999a
). To determine the effect of IL-5, IL-6, and IL-10 deficiencies on vaccine protection from acute virus infection, vaccinated and challenged mice were assayed for spleen infectious centres at 2 weeks post challenge. Most vaccinated B6 mice were protected from spleen infectious centres after FV inoculation (Fig. 2
). Results from the vaccinated cytokine-deficient mice were quite similar to wild-type mice with very low or no spleen infectious centres (Fig. 2
). Thus, IL-5, IL-6 and IL-10 did not appear essential for the vaccine-induced protection elicited by infection with live attenuated virus.
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In contrast to IL-5, IL-6 has been reported to play a significant role in immunity to several virus infections. Our findings that secondary antibody responses against FV were impaired in IL-6-/- mice are similar to previous reports showing reduced humoral immune responses to vesicular stomatitis virus, vaccinia virus (VV) and influenza virus in IL-6-deficient mice (Kopf et al., 1994 ; Ramsay et al., 1994
). The cellular immunity of IL-6-/- mice was found to be reduced after VV but not after lymphocytic choriomeningitis virus infection (Kopf et al., 1994
). The impaired antiviral immune responses led to a severely compromised ability of IL-6-/- mice to clear acute infections with VV, ectromelia virus or murine cytomegalovirus (Ramshaw et al., 1997
). Interestingly, although IL-6-/- mice were also more susceptible to herpes simplex virus type 1-induced disease (LeBlanc et al., 1999
) and showed significantly enhanced virus replication after acute FV infection (Fig. 1
), the control of persistent virus was not affected in either model. Thus, the proinflammatory cytokine IL-6 has important immune regulatory functions during acute viral infections but does not seem to play a significant role in the immunological control of persistent viruses. In all studies with IL-6-deficient mice it has to be taken into account that the lack of IL-6 affects lymphoid proliferation in vivo leading to a 2040% reduction of T-cell numbers in comparison with wild-type mice (Kopf et al., 1994
). Thus, some of the impairments in viral defence mechanisms of IL-6-/- mice might in part be due to lower T-cell numbers.
IL-10 is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage and T-cell functions (Moore et al., 1993 ). In support of these findings, IL-10-/- mice often develop a chronic bowel inflammation due to the lack of immunosuppressive IL-10 (Kuhn et al., 1993
). However, our results and a study with human papillomavirus (Santin et al., 2000
) challenge the view of IL-10 as an exclusively immunosuppressive cytokine. During acute FV infection IL-10 was obviously important for restriction of virus replication in vivo (Fig. 1
). Santin et al. (2000)
and others (Chen & Zlotnik, 1991
) have described that IL-10 can act as a differentiation factor for CD8+ cytotoxic T-lymphocytes (CTL). Since CD8+ T cells play an important role in immunity of mice against FV (Robertson et al., 1992
), reduced CTL activity in IL-10-/- mice could be a likely explanation for the enhanced FV load in the spleens of these mice. In contrast to our findings, the infection of IL-10-/- mice with mouse hepatitis virus did not lead to higher virus loads compared than wild-type mice (Lin et al., 1998
). This indicates that the involvement of IL-10 in antiviral immune responses is not a general phenomenon, but relevant only in certain virus infections.
The current results establish an important function for IL-6 and IL-10 in restriction of an acute retroviral infection. However, both these cytokines as well as IL-5 seem to be dispensable for vaccine-induced protective immunity against FV.
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Acknowledgments |
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References |
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Received 15 September 2000;
accepted 29 January 2001.