Anneline Nansen

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell‐independent encephalitis, and infection resulted in a dramatic early up‐regulation of chemokine gene expression. Similar marked up‐regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV‐infected mice, only LCMV‐induced T cell‐dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus‐activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+‐dependent CNS inflammation.

Efficient T-cell surveillance of the CNS requires expression of the CXC chemokine receptor 3

T-cells play an important role in controlling viral infections inside the CNS. To study the role of the chemokine receptor CXCR3 in the migration and positioning of virus-specific effector T-cells within the brain, CXCR3-deficient mice were infected intracerebrally with lymphocytic choriomeningitis virus (LCMV). Analysis of the induction phase of the antiviral CD8+ T-cell response did not reveal any immune defects in CXCR3-deficient mice. Yet, when mice were challenged with LCMV intracerebrally, most CXCR3-deficient mice survived the infection, whereas wild-type mice invariably died from CD8+ T-cell-mediated immunopathology. Quantitative analysis of the cellular infiltrate in CSF of infected mice revealed modest, if any, decrease in the number of mononuclear cells recruited to the meninges in the absence of CXCR3. However, immunohistological analysis disclosed a striking impairment of CD8+ T-cells from CXCR3-deficient mice to migrate from the meninges into the outer layers of the brain parenchyma despite similar localization of virus-infected target cells. Reconstitution of CXCR3-deficient mice with wild-type CD8+ T-cells completely restored susceptibility to LCMV-induced meningitis. Thus, taken together, our results strongly point to a critical role for CXCR3 in the positioning of effector T-cells at sites of viral inflammation in the brain.

Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.

The expression of major histocompatibility class I (MHC‐I) crucially depends upon the binding of appropriate peptides. MHC‐I from natural sources are therefore always preoccupied with peptidescomplicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC‐I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC‐I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC‐I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC‐I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre‐oxidized recombinant MHC‐I.

Sensitization to Lipopolysaccharide in Mice with Asymptomatic Viral Infection: Role of T Cell-Dependent Production of Interferon-γ

The interplay between viral infection and lipopolysaccharide (LPS) was studied. Infection with a noncytopathogenic virus, lymphocytic choriomeningitis virus (LCMV), was found to sensitize mice to low doses of LPS. In vivo, this hypersensitivity correlated with hyperproduction of tumor necrosis factor-alpha (TNF-alpha), and in vitro, LPS-stimulated splenic adherent cells produced increased amounts of TNF-alpha. Hyperproduction of TNF-alpha was temporally correlated with virus-induced production of interferon-gamma (IFN-gamma); only marginally increased IFN-gamma and TNF-alpha production was observed in LCMV-infected, T cell-deficient mice and in mice infected with vesicular stomatitis virus, a virus that induces much less T cell activation than does LCMV. Finally, LCMV infection was much less efficient in priming IFN-gamma knockout mice for hyperproduction of TNF-alpha. These findings indicate that clinically silent viral infections may induce hypersensitivity to LPS through T cell activation and subsequent production of IFN-gamma; this sensitizes monocytes/macrophages for hyperproduction of TNF-alpha.

Viral Infection Causes Rapid Sensitization to Lipopolysaccharide: Central Role of IFN-αβ

LPS is the major active agent in the pathogenesis of Gram-negative septic shock. In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock. We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection. Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect. This hypersensitivity to LPS correlated with hyperproduction of TNF-α in vivo. The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice. Our results revealed that while NK cell depletion and elimination of IFN-γ partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-αβR-deficient mice. Thus hyperproduction of TNF-α was completely abrogated in IFN-αβR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-αβ-mediated priming of mice for an augmented production of TNF-α in response to LPS. This conclusion was further supported by the finding that pretreatment of mice with rIFN-αβ mimicked the effect of viral infection. In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-αβ and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection.

Opposing Effects of CXCR3 and CCR5 Deficiency on CD8+ T Cell-Mediated Inflammation in the Central Nervous System of Virus-Infected Mice

T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice were generated and infected intracerebrally with noncytolytic lymphocytic choriomeningitis virus. Because these chemokine receptors are mostly expressed by overlapping subsets of activated CD8+ T cells, it was expected that absence of both receptors would synergistically impair effector T cell invasion and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CXCR3/CCR5-deficient mice, more CD8+ T cells were found in the parenchyma of double-deficient mice when these were analyzed around the time when the difference in clinical outcome becomes manifest. Taken together, these results indicate that while CXCR3 plays an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response.

Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines

T cell mediated immunity and in particular CD8+ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8+ T cells to sites of viral infection.

The Virus-Encoded Chemokine vMIP-II Inhibits Virus-Induced Tc1-Driven Inflammation

ABSTRACT The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+ T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+ T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.

Cytokine vaccination: neutralising IL-1α autoantibodies induced by immunisation with homologous IL-1α

Abstract High-affinity IgG autoantibodies (aAb) to IL-1α are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1α, we immunised mice with recombinant murine IL-1α. Unprimed and Bacille Calmette-Guerin (BCG)-primed BALB/cA mice were vaccinated with IL-1α coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1α. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1β. The induced anti-IL-1α aAb inhibited binding of IL-1α to the murine T-cell line NOB-1 by simple competition and neutralised IL-1α, but not IL-1β-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1α can be induced in mice by vaccination with recombinant murine IL-1α conjugated to PPD. Studies of the effects of IL-1α aAb in such animals may help clarify the importance of naturally occurring IL-1α aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-gamma-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.