Footpad inoculation of naive mice with a recombinant TK+ ECTV expressing mouse IL-4 results in systemic infection and suppression of splenic NK and CTL cytolytic activity and IFN-γ expression. The expression of IL-4 by ECTV renders the virus lethal to mice that are normally genetically resistant. Importantly, memory responses in mice previously immunized with ECTV were also inhibited, leading to uncontrolled viral replication in the visceral organs and resulting in classic symptoms of acute mousepox.
Previous studies using the closely related orthopoxvirus VACV have established that IL-4 coexpression is associated with a delay in virus clearance from the major target organs (2
). This is likely to be the result of the observed combined reduction in antiviral effectors such as CD8+
CTL precursor (CTLp) development, IFN-γ expression, macrophage activation, and inducible nitric oxide production (4
). While no reduction in splenic NK cell activity was observed in infected CBA mice (41
), a significant reduction was found when SCID mice were infected with VACV expressing IL-4 (4
). It is clear from the VACV model that IL-4 down-regulates in vivo expression of the key type 1 cytokines IL-2, IL-12, and IFN-γ (41
), which are pivotal for stimulation and differentiation of antiviral cell-mediated effectors such as NK, CD8+
CTL, and CD4+
Th1 cells (3
In the classical ECTV pathogenesis study conducted by Fenner (12
), replicating wild-type Moscow strain of ECTV can first be detected in the liver and spleens of mice by 3 days p.i. At this stage in the present study, virus-encoded IL-4 had a significant effect on the cytolytic activity of NK cells in these organs. The observed partial down-regulation of NK cell activity seen here is likely to be the result of the combined IL-4-induced suppression of IL-12 expression by antigen-presenting cells (7
) and type 1 cytokine receptors on NK cells (30
) inhibiting activation and proliferation. In the VACV IL-4 infection model, IL-12 expression in the spleen was clearly down-regulated by day 2 p.i. (41
). The remaining NK cell cytolytic activity observed during infection with ECTV-IL4(TK+) probably results from the more usual mode of IFN-α/β induction generally seen during viral infections (3
Later, at day 7 p.i., when the adaptive anti-ECTV cell-mediated response in the spleen should be near maximal (29
), there was no measurable CD8+
cytolytic activity or IFN-γ expression by isolated splenocytes of mice infected with ECTV-IL4(TK+). Viral replication appeared to be uncontrolled, with the mice dying shortly thereafter. This is consistent with the IL-4-induced suppression of IL-12/IFN-γ expression inhibiting CTLp development (41
). Under some circumstances, stimulation of naive CD8+
cells in the presence of IL-4 can generate noncytotoxic Tc2 cells (10
). In addition, in vitro IL-4 treatment of activated Tc1 cells has been shown to cause defective cytokine expression and inhibit proliferation in response to antigen stimulation (37
). Abnormally high IL-4 levels, accompanied by suppressed production of IL-12 and IFN-γ, could potentially result in the generation of either Tc2 cells with reduced cytolytic activity or defective Tc1 cells, which may also account for the observed reduced CD8+
lytic activity and IFN-γ expression following infection with ECTV-IL4(TK+). In marked contrast to the VACV IL-4 model, however, infection with ECTV-IL4(TK+) and suppression of CD8+
effector function appears to be absolute, which could be a consequence of the greater ability of ECTV to replicate in the mouse, with higher and sustained levels of IL-4 expression compared to VACV.
It is also possible that high levels of ECTV-IL4(TK+) replication in the spleen may have resulted in enhanced lymphocyte killing, accounting for the observed reduction in cytotoxic activity and IFN-γ expression. However, IL-4 expression by poxviruses is not known to confer increased replicative ability in vivo. Indeed, peak titers of VACV-IL4 in the ovary (41
) and spleen (2
) or of attenuated ECTV-IL4 at the inoculation site (C. D. Christensen, R. J. Jackson, and A. J. Ramsay, unpublished results) are equivalent to those seen in infection with control virus.
A similarly constructed TK−
ECTV expressing IL-4 was attenuated upon infection of the naive CBA mice, and replication of this virus was restricted to the inoculated footpad, accompanied by extreme swelling (Christensen et al., unpublished). More importantly, there was no measurable reduction in the generation of splenic cytolytic lymphocyte or IFN-γ responses compared to control virus infection, although clearance of the virus from the inoculated foot was again considerably delayed. This suggests that the induced suppression of the antiviral NK, CTL, and IFN-γ responses observed in the present study was localized to the site of viral expressed IL-4 in the lymphoid tissue. It has previously been shown that the TK+
phenotype of ECTV is required for replication in macrophages, allowing dissemination from the site of inoculation and viral replication in the liver and spleen (26
). In the present study, dissemination of ECTV-IL4(TK+) to the visceral organs, followed by systemic expression of IL-4, suppressed development of cell-mediated cytotoxic responses in the lymphoid tissues, culminating in uncontrolled viral replication, acute organ failure, and death.
It is clear from these studies that IL-4 expression also permits uncontrolled viral replication in the visceral organs of ECTV-immune mice, indicating that this factor can inhibit the generation of effector cells from a pool of memory T cells. In contrast, preexisting immunity to ECTV was sufficient to limit reinfection with either control or virulent Moscow virus. T-cell immunological memory is considered to result from enhanced numbers of antigen-specific CTLp and residual populations of activated CTL effector cells (9
). Thus, even in the presence of preexisting immunity, IL-4 can inhibit the expression of immunological memory. These findings demonstrate the effectiveness of IL-4 for the inhibition of powerful cell-mediated immune reactions and suggest strategies potentially useful for the control of deleterious immune responses, such as autoimmune reactions.