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Journal of Virology, June 2005, p. 7768-7776, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7768-7776.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Indoleamine 2,3-Dioxygenase Mediates Cell Type-Specific Anti-Measles Virus Activity of Gamma Interferon

Karola Obojes,¹ Oliver Andres,¹ Kwang Sik Kim,² Walter Däubener,³ and Jürgen Schneider-Schaulies^1*

Institut für Virologie und Immunbiologie, Julius-Maximilians-Universität, 97078 Würzburg, Germany,¹ Johns Hopkins University School of Medicine, Baltimore, Maryland 21287,² Institut für Medizinische Mikrobiologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany³

Received 19 July 2004/ Accepted 26 January 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Gamma interferon (IFN-) has been shown to be increased in sera from patients with acute measles and after vaccination, to exhibit protective functions in brains of patients with subacute sclerosing panencephalitis, and to mediate a noncytolytic clearance of measles virus (MV) from rodent brains. In order to reveal a possible intracellular antiviral activity in the absence of antigen presentation and cytotoxic T cells, we investigated IFN--induced effects on MV replication in various tissue culture cells. While attenuated MV strains are more sensitive to IFN-/ß than are wild-type strains, IFN- inhibits the replication of all MV strains in epithelial, endothelial, and astroglial cells, but not in lymphoid and neuronal cell lines. The antiviral activity induced by IFN- correlates with the induction of indoleamine 2,3-dioxygenase (IDO), an enzyme of the tryptophan degradation pathway known to mediate antiviral as well as antibacterial and antiparasitic effects. The IFN--induced antiviral activity can be overcome by the addition of excess amounts of L-tryptophan, which indicates a specific role of IDO in the anti-MV activity. Our data suggest that the IFN--induced enzyme IDO plays an important antiviral role in MV infections of epithelial, endothelial, and astroglial cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alpha/beta and gamma interferons (IFN-/ß and IFN-, respectively) play a major role in the antiviral defense of the innate and adaptive immune system. The antiviral effects of interferons can be direct (intracellular) and indirect, involving effector cells of the immune system (11). It is known that IFN-, one of the T-helper 1-type cytokines, induces an intracellular activity against several viruses, including herpes simplex virus (10, 48), human parainfluenza virus (12), mouse hepatitis virus (45), hepatitis C virus (20), Sindbis virus (6), vaccinia virus (30), and vesicular stomatitis virus (29).

Although the mechanism of action is not known, IFN- is supposed to also play an important role against measles virus (MV) in acute and persistent infections. After acute infections and vaccinations, IFN- concentrations in the serum are increased (41, 43). This cytokine can also be detected in brain lesions of patients suffering from subacute sclerosing panencephalitis (SSPE) (38), a complication developing years after acute MV infection on the basis of a persistent infection of the brain (51, 58). Interestingly, the peripheral blood mononuclear cells of most SSPE patients have a reduced capacity to respond to MV infection by producing IFN- (27). When SSPE patients were divided into responders (group A) and nonresponders (group B) according to their IFN- response to the infection, all patients in group A retained cognitive function for a long time, while most patients in group B lost this function rapidly (27). This indicates that IFN- exerts an important antiviral function in MV infections in humans.

The importance of IFN- as a mediator of the anti-MV defense has been confirmed with a rodent model of experimentally induced encephalitis. IFN--depleted adult BALB/c mice become susceptible to the infection and die after 6 to 15 days (19). Upon the neutralization of IFN- with antibodies in vivo, the phenotype of MV-specific T-helper cells isolated from BALB/c mice is reversed from subtype 1 to 2 (19). Furthermore, the neutralization of IFN- interferes with major histocompatibility complex class II-dependent antigen presentation and the subsequent proliferation of CD4⁺ T cells in vitro and in vivo (61). The reduction in numbers of CD4⁺ T cells below a protective threshold may lead to susceptibility to MV-induced encephalitis. From these results, however, it was not clear whether the antiviral effect of IFN- is exerted only indirectly, via antigen presentation and effector cells of the immune system, or also directly, via intracellular mechanisms. The use of transgenic mice lacking CD4⁺ cells, ß-2 microglobulin (CD8⁺ cells), the pore-forming protein perforin, or IFN- revealed that IFN- in the absence of immune effector cells can cause a noncytolytic elimination of virus not only from the brain, but also from murine neurons in tissue culture (46). These data suggest that in addition to its effects on the adaptive immune system, IFN- can induce an intracellular activity against MV.

A variety of IFN--regulated antiviral mechanisms may be induced in target cells. The most prominent antiviral mechanism may be the induction of nitric oxide synthase (iNOS), NO, and reactive oxygen products. In addition, one well-documented cellular response to IFN- is the induction of a considerable and sustained tryptophan catabolism influencing the immune response and the replication of pathogens (for a review, see reference 35). The molecular basis leading to growth inhibition of parasites, bacteria, and viruses is not well understood, and two theories (the tryptophan depletion and tryptophan utilization theories) at present cannot satisfactorily explain the observed effects. The enzyme indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step in the kynurenine pathway of tryptophan degradation, plays a central role in the conversion of tryptophan to N-formyl-kynurenine. Strong IDO activity leads to a nearly complete depletion of the essential amino acid tryptophan at the local site of inflammation. This may result in growth arrest of the involved pathogen but also in the suppression of T-cell responses. Suppression of the T-cell response was also found after the expression of IDO in dendritic cells, in mice with IDO-expressing tumors leading to an escape from cytotoxic responses, and after the expression of IDO in trophoblasts as a mechanism preventing the immune response against fetal tissue during gestation (21, 34, 36, 37, 59, 60). Furthermore, IDO activation results in the generation of toxic metabolites produced within the kynurenine pathway, e.g., N-formyl-kynurenine, 3-hydroxy-kynurenine, and antranilic acid (62). Thus, the role of IDO in vivo is ambivalent and may depend on additional factors that are present during specific immune responses.

The antiparasitic, antibacterial, and antiviral activities of IFN- correlate with the induction of IDO (25, 26). It has been demonstrated that IDO mediates the activity of IFN- against viruses such as human cytomegalovirus (CMV) (7) and herpes simplex virus type 1 (HSV-1) (3). For these infections, the specificity of the IDO action was demonstrated by adding excess amounts of L-tryptophan, which reverts the antiviral effect of IFN-. We describe here the antiviral effect of IFN--induced IDO on MV replication.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokines, antibodies, and cells. Recombinant human IFN- was purchased from Tebu-Biotec, and recombinant human IFN-, interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-) were purchased from Strathmann Biotec. A mouse monoclonal anti-MV nucleocapsid (N) antibody was produced from the F227 hybridoma, using RPMI medium containing 10% fetal calf serum (FCS), and purified with protein G-Sepharose in our laboratory. We used Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (Molecular Probes) as a secondary antibody. Cell nuclei were stained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) nucleic acid stain (Molecular Probes).

Primary human umbilical vein endothelial cells (HUVEC) were prepared from umbilical cords obtained from the maternity ward of the University Hospital, Würzburg, Germany, as described previously (5, 33). HUVEC were cultivated in M199 medium (Gibco) containing 25 mM HEPES, 20% FCS (Biochrom), 5 U/ml heparin, 30 µg/ml endothelial cell growth supplement (Sigma), and 100 U/ml penicillin-streptomycin. Simian virus 40 large T antigen-transformed human brain microvascular endothelial cells (HBMEC) (5, 55) were grown in RPMI 1640 medium (Gibco) containing 25 mM HEPES, GlutaMAX I (Gibco), 10% FCS (Biochrom), 10% NuSerum IV (Becton Dickinson), 1x nonessential amino acids and vitamins, 1 mM sodium pyruvate, 5 U/ml heparin, 30 µg/ml endothelial cell growth supplement (Sigma), and 100 U/ml penicillin-streptomycin. For both endothelial cell types, the surfaces of plastic dishes were coated with 0.5% gelatin (Sigma). Human neuroblastoma IMR-32 cells were grown in RPMI 1640 medium containing 20% FCS and 1% nonessential amino acids, SKN-MC and NT2 cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS and 1x minimum essential medium (MEM) with nonessential amino acids, astrocytoma 86HG39 cells (3) were grown in MEM containing 10% FCS, the Epstein-Barr virus-transformed B-cell line BJAB was grown in RPMI 1640 containing 10% FCS, human lung carcinoma A549 cells were grown in Ham's F-12 nutrient medium containing 10% FCS, and Vero cells were grown in MEM containing 5% FCS.

Measles viruses. The MV wild-type strain Wü4797 (Würzburg.DEU/96/4797) was grown on BJAB cells. The vaccine-like MV Edm strain (Edmonston B) and an Edm-based recombinant MV expressing enhanced green fluorescent protein (GFP) from its first open reading frame (EdmGFP; a kind gift of P. Duprex, Belfast, United Kingdom) (16) were propagated on Vero cells. For virus propagation, cells were infected at a multiplicity of infection (MOI) of 0.001, and viruses were harvested when maximal giant cell formation was observed. To determine virus replication in the absence or presence of interferons, we infected cells at an MOI of 1 for single-step growth analysis or 0.1 for experiments investigating the susceptibility to tryptophan and harvested the total amount of virus after the indicated times by one cycle of freeze-thawing of cells plus supernatant and by twice pelleting the cell debris by centrifugation. Supernatants were stored at –80°C. The amount of newly synthesized virus was quantified by a plaque assay using Vero cells (for the vaccine virus) or Vero-hSLAM cells (for the wild-type virus) (a kind gift of Y. Yanagi [42]). Statistical analyses were done by using the program Prism (unpaired t test for two-tailed P values).

N-formyl-kynurenine assay. The activity of IDO is strictly correlated with the concentration of N-formyl-kynurenine in supernatants of tissue culture cells, which allows the measurement of its activity in supernatants (15, 57). The activity of IDO was determined as described previously (15). Briefly, cells (3 x 10⁴ per well) were plated in 96-well flat-bottomed microtiter plates in which the first three wells contained 1,000 U/ml IFN- in 200 µl Iscove's modified Dulbecco's medium containing 5% FCS (Cambrex) and the following triplicates contained twofold dilutions of the cytokine and medium as controls. All wells contained an additional externally added 100 µg/ml L-tryptophan (L-Trp; Merck). The plates were incubated for 72 h at 37°C, and thereafter 160 µl was removed from each well and transferred to a 96-well V-bottomed plate. After the addition of 10 µl of 30% trichloroacetic acid (Merck) to each well, the plates were incubated for 30 min at 50°C to hydrolyze N-formyl-kynurenine to kynurenine. After centrifugation for 10 min, 100 µl of supernatant was transferred to a 96-well flat-bottomed plate and mixed with 100 µl of fresh 1.2% 4-(dimethylamino)benzaldehyde (Ehrlich reagent; Sigma), and 10 min later the absorbance at 492 nm was determined with a microplate reader (Tecan). Data were measured as mean optical densities of triplicate cultures, and the concentration of kynurenine was calculated according to a standard curve of L-kynurenine sulfate (Sigma). In cases of induction of IDO by viruses, cells were incubated with IFN- and an externally added 100 µg/ml L-tryptophan for 3 days and then with virus and 100 µg/ml L-tryptophan for an additional 3 days before the kynurenine concentration was determined.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular IFN-/ß- and IFN--induced anti-measles virus activities. It was previously reported that the antiviral effect of IFN-/ß is MV strain specific. Wild-type MV has the capacity to suppress the induction as well as the inhibitory effect of IFN-/ß, whereas the vaccine virus is susceptible to antiviral activities (39). Using this as a control, we observed considerably less virus spread in the presence of IFN- when human brain microvascular endothelial cells (HBMEC) were infected with the vaccine strain of MV (strain Edm; Fig. 1, compare panels A and B) than that after infection with wild-type MV (Wü4797; Fig. 1, compare panels D and E). In contrast, IFN- treatment of endothelial cells reduced the syncytium formation of both the vaccine strain and wild-type MV (Fig. 1, compare panels A and C and panels D and F). The magnifications (Fig. 1A' to F') in the figure show morphological differences in the nucleocapsid complexes observed in infected cells. IFN- treatment reduced the plaque size and induced the formation of small dot-like nucleocapsid complexes in endothelial cells. Virus yields from such cultures (infected at an MOI of 0.1) on day 3 postinfection were as follows for the vaccine strain: 9.0 x 10⁵ ± 1.4 x 10⁵ in the absence of IFN-, 9.2 x 10⁴ ± 1.1 x 10⁴ in the presence of IFN-, and 5.7 x 10⁴ ± 1.1 x 10⁴ in the presence of IFN-. In the case of the wild-type virus, the yields were 1.2 x 10⁵ ± 0.1 x 10⁵ in the absence of IFN-, 1.1 x 10⁵ ± 0.1 x 10⁵ in the presence of IFN-, and 3.6 x 10⁴ ± 0.6 x 10⁴ in the presence of IFN- (n = 3; P values were <0.001).

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FIG. 1. Differential effects of interferons on morphology of MV-infected cultures of endothelial and lymphoid cells. HBMEC (A to F) and BJAB cells (G to O) were infected with the MV vaccine (Edm) or wild-type (Wü4797) virus at an MOI of 0.1 in the absence or presence of 500 U/ml IFN- or IFN-, as indicated. Infected HBMEC were fixed and permeabilized after 48 h, and viral N proteins were stained with the monoclonal antibody F227 and with secondary Alexa Fluor 488-conjugated antibodies (green). Cell nuclei were counterstained with DAPI (blue). The differential cytopathic effects are shown at higher magnifications (A' to F'). Bar, 100 µm (C) or 20 µm (C'). Fully developed cytopathic effects can be seen in panels A', D', and E', whereas the extension of the nucleocapsid-positive plaque is restricted in B', and even smaller and dot-like nucleocapsid complexes are observed in C' and F'. Phase-contrast pictures are shown in the case of BJAB cells (G to O). Panels G to O are given at the same magnification (bar, 100 µm). Large fused giant cells and cell aggregates can be seen in wild-type-infected cultures (M to O), whereas the vaccine virus induced smaller giant cells (J to L).

For the human transformed B-cell line BJAB, considerably smaller giant cells were observed after infection with the MV vaccine strain than with the wild-type virus (Fig. 1J and M). The addition of IFN- slightly reduced the syncytium size of vaccine virus-infected cells (Fig. 1K) but not that of wild-type-infected cells (Fig. 1N). The addition of IFN- had no effect on giant cell formation after the infection of lymphoid cells with either type of virus (Fig. 1L and O). Virus yields from such BJAB cultures were as follows for the vaccine virus: 9.9 x 10⁵ ± 0.1 x 10⁵ in the absence of IFN-, 2.9 x 10⁵ ± 1.9 x 10⁵ in the presence of IFN-, and 8.7 x 10⁵ ± 0.3 x 10⁵ in the presence of IFN-. In the case of the wild-type virus, the yields were 1.3 x 10⁵ ± 0.1 x 10⁵ in the absence of IFN-, 8.7 x 10⁴ ± 0.4 x 10⁴ in the presence of IFN-, and 9.7 x 10⁴ ± 1.1 x 10⁴ in the presence of IFN- (n = 3; P values were <0.001). This shows that IFN- can induce antiviral activity against both the vaccine strain and wild-type MV, but only in certain susceptible cells such as HBMEC.

To quantify the effects of interferons on virus replication in various cells, we titrated newly synthesized MV-Edm in the presence or absence of IFN-, and as a control, also in the presence of IFN- (Fig. 2) (100 U/ml of both cytokines). IFN- induced strong antiviral activities in human epithelial and endothelial cells (Fig. 2A to C). In epithelial A549 cells and primary human umbilical vein endothelial cells (HUVEC), IFN- exerted the strongest effects, reducing viral titers approximately 100-fold. In HBMEC, the replication of MV was inhibited by IFN-, by a factor of approximately 1 log. Similar results were obtained with African green monkey kidney epithelial cells (Vero cells) and human astroglioma 86HG39 cells (not shown). Using a similar concentration of IFN- (100 U/ml) as a control, we detected antiviral effects of IFN-/ß, with viral titers reduced 5- to 10-fold. In contrast to the case for epithelial, endothelial, and astroglial cells, IFN- and IFN- did not inhibit MV infection of human neuroblastoma IMR-32 cells (Fig. 2 D) or of the human B-cell line BJAB (not shown). These findings demonstrate that IFN- induces an intracellular activity against MV specifically in certain cells.

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FIG. 2. Effect of IFN- and IFN- on MV replication in various cell types. Human epithelial A549 cells (A), HUVEC and transformed HBMEC (B and C, respectively), and neuroblastoma IMR-32 cells (D) were infected at an MOI of 1 with MV-Edm in the absence or presence of 100 U/ml IFN- or 100 U/ml IFN-. After 1 to 5 days, the amounts of newly synthesized virus (cell bound and released together) were determined by a plaque assay (n = 4; data are from four independent assays and include standard deviations). Virus titers are presented as circles (medium control), squares (IFN-), and triangles (IFN-). The inhibition of virus growth by IFN- was statistically highly significant, with P values of <0.0001 (A and B) and <0.0005 (C) on day 3 (unpaired t test). In the case of IMR-32 cells, the P value on day 3 was 0.49 (D).

In order to investigate whether the induction of the antiviral state may require more time, we pretreated cells for 24 h with IFN- or IFN- before infection with MV. In A549 cells and HBMEC, the pretreatment of cells led to a significant but small enhancement of the antiviral effect compared to the case after the addition of IFN- together with the virus (Fig. 3A and B). The addition of 100 U/ml TNF-, used as a control, did not affect MV replication. In IMR-32 and BJAB cells, the pretreatment also did not induce antiviral activity (not shown). Thus, antiviral mechanisms are rapidly induced by IFN- in susceptible cells.

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FIG. 3. Effect of IFN- preincubation on antiviral activity and on the growth of cell lines. A549 cells (A) and HBMEC (B) were treated with 100 U/ml IFN-, IFN-, and TNF- (as a control) either 24 h before infection at an MOI of 1 and during the infection period (white columns) or only during the time of infection (black columns). Cells were infected with a recombinant MV expressing GFP (EdmGFP), and the autofluorescence of the cultures was quantified with a fluorescence reader (Labsystems) at 48 h postinfection. The data are expressed as percentages of the fluorescence of the medium control (100%). Experiments were performed in triplicate, and the mean values and standard deviations were calculated. The differences between virus replication in IFN--pretreated and nonpretreated cells were significant for A549 cells and HBMEC, with P values as indicated (unpaired t test). The effect of IFN- on the cell number was determined in the absence and presence of 100 and 1,000 U/ml IFN- for A549 (C) and IMR-32 (D) cells. Trypan blue-including and -excluding cells were counted microscopically, and numbers of living cells are given.

As an additional control, we measured the effect of IFN- on cell growth and survival. IFN- treatment induced a transient delay in the proliferation of susceptible cells, such as A549 cells (Fig. 3C) and HBMEC (not shown), whereas in cells refractory to IFN-, such as IMR-32 (Fig. 3D) and BJAB (not shown) cells, no impact on the growth rate was detected. The percentages of dead cells in these cultures, as determined microscopically by trypan blue exclusion, were not significantly altered by the addition of IFN- and were 3.1% for A549 cells, 1.8% for IMR-32 cells, 1.5% for HBMEC, and 0.5% for BJAB cells with 100 U/ml IFN- and between 4.3 and 3.1% with 1,000 U/ml IFN-. Thus, cell growth and survival are not strongly affected by IFN-, whereas virus growth is specifically restricted in certain cells.

Correlation of antiviral IFN- effect and induction of IDO activity. Since IDO is one of the IFN--induced enzymes with antiviral activity, we investigated the capacity of IFN- to induce IDO in the cells used in this study. IDO activity was measured by the detection of kynurenine in the supernatants of cells treated with increasing amounts of IFN- alone or in combination with IL-1ß, which can enhance IDO activity. A high level of IDO activity following IFN- treatment was detected in A549 cells and HBMEC (Fig. 4A and B), whereas no IDO activity was found in BJAB and IMR-32 cells (Fig. 4 C and D) or in the neuroblastoma cell lines SKN-MC and NT2 (not shown). IL-1ß (100 U/ml) in combination with IFN- led to a selective superinduction of IDO in cells in which IDO activity was induced by IFN-. No IDO activity was detected in cells treated with IL-1ß alone. The data indicate that IDO induction correlates with the antiviral activity induced by IFN- and thus may contribute to the activity against MV in epithelial and endothelial cells.

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FIG. 4. Cell specificity of IDO induction. Cells were treated with IFN- at increasing concentrations, as indicated, in the absence and presence of IL-1ß. The specifically IDO-dependent N-formyl-kynurenine synthesis was determined by the quantification of kynurenine in the cell supernatant with Ehrlich reagent as described in Materials and Methods. The amounts of kynurenine production after 3 days in culture are given in µg/ml, calculated according to the optical densities of the kynurenine standards for epithelial A549 cells (A), HBMEC (B), lymphoid BJAB cells (C), and neuronal IMR-32 cells (D). In cells in which IDO was inducible by IFN-, the addition of 100 U/ml IL-1ß led to a superinduction of enzyme activity. The data were determined as triplicates in three independent experiments (n = 3).

Influence of viral infection on kynurenine production. Since viral infections may contribute to the induction of IDO, as described for macrophages (22, 23), we investigated whether MV infection itself may induce IDO or may influence IDO induction by IFN-. The IDO activities in A549 cells and HBMEC in the presence of increasing concentrations of IFN- were determined for uninfected and MV-Edm-infected cells (Fig. 5A and B). Cells were treated with IFN- for 3 days and subsequently infected for 3 days before the kynurenine concentration in the supernatant was determined. For both cell lines, the infection alone did not induce IDO activity (Fig. 5, U/ml IFN-). For A549 cells, there was a considerable enhancing effect of the infection on IFN--induced kynurenine production. However, for HBMEC, there was no significant effect of MV infection on the IFN--induced IDO activity.

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FIG. 5. IDO induction in the absence and presence of viral infection. The IDO activity in A549 cells (A) and HBMEC (B) in the presence of increasing concentrations of IFN- for 3 days and after an additional 3 days of culture in the absence or presence of virus (MV-Edm; MOI = 0.1) was determined as described in Materials and Methods. The amounts of kynurenine production are given in µg/ml, calculated according to the optical densities of the kynurenine standards. The data are triplicates with standard deviations from one representative experiment of three independent experiments. The increase in the kynurenine concentration in infected A549 cells was significant, with P values of <0.0001 with 125 and 250 U/ml IFN- (unpaired t test).

The results shown in Fig. 4 and 5 cannot be directly compared, since for the data shown in Fig. 5 cells were pretreated for 3 days in the presence of 100 µg/ml L-tryptophan and then infected and incubated for an additional period of 3 days, with a newly added 100 µg/ml L-tryptophan in the medium. This resulted in higher concentrations of newly synthesized kynurenine in the supernatants.

Abrogation of antiviral IFN- effect by tryptophan. IDO-induced antiviral effects can be specifically blocked by the addition of excess amounts of tryptophan to the medium of cultured cells (57). To demonstrate the specific contribution of IDO to the IFN--induced antiviral effects, we added L-tryptophan to infected cultures. The IFN- effect was abrogated by the addition of 100 µg/ml L-tryptophan to the medium and increased virus titers 20- to 30-fold in A549 cells and approximately 10-fold in HBMEC (Fig. 6A and B). In cells treated with IFN- and IL-1ß in combination, which led to an overinduction of IDO as shown in Fig. 4, L-tryptophan restored viral titers to similar levels as those in cells treated with IFN- alone (Fig. 6C and D). In the absence of IFN-, the addition of L-tryptophan had no effect on virus titers (Fig. 6A to D, U/ml IFN-).

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FIG. 6. Abrogation of the IFN--induced antiviral effect by the addition of L-tryptophan. A549 cells (A and C) and HBMEC (B and D) were infected with MV-Edm (MOI = 0.1) in medium containing increasing concentrations of IFN- without (A and B) and with (C and D) 100 U/ml IL-1ß in the absence (white columns) or presence (black columns) of 100 µg/ml L-tryptophan. After 48 h, the amounts of newly synthesized virus were quantified by titration and are given as PFU/ml. The data are mean values from three experiments (duplicates). The differences between controls (0 U/ml IFN-) and IFN--treated titers were highly significant, with P values of <0.003 (unpaired t test). The differences between titers with and without tryptophan were also highly significant, with P values of <0.006. Examples of P values are given in the figure.

For all cells, the abrogation of the IFN--induced antiviral effect was not complete, suggesting that other IFN--induced mechanisms besides IDO also contribute to the effect. As a control, we added L-tryptophan to cells that had been treated with IFN-. In these cells, the antiviral activity was not abrogated (not shown). Overall, our results indicate that the IFN--induced anti-MV activity in endothelial and epithelial cells is largely mediated by the tryptophan-degrading enzyme IDO.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we describe that IFN- induces an intracellular activity against MV in endothelial, epithelial, and astroglial cells. It was observed earlier that IFN-/ß exerts activity against attenuated MV strains, whereas wild-type isolates have the capacity to counteract the IFN-/ß response by suppression of the induction of interferon and by blocking IFN-/ß-induced intracellular activities. The V and C proteins of MV are involved in these processes (39, 47, 54) by interacting with STAT proteins and IRF9 (44, 56, 63). We observed the same differences between vaccine and wild-type MV strains with IFN--treated cells. In contrast to the case with IFN-/ß, we found that IFN- induces an intracellular activity against MV, irrespective of which strain of virus was used. This and the finding that L-tryptophan cannot abrogate the effect of IFN-/ß indicate the differences between the two antiviral mechanisms. The antiviral activity induced by IFN- correlates with the inducibility of IDO activity. Since the specificity of the reaction was demonstrated by the abrogation of the effect with excess L-tryptophan, we concluded that IDO mediates the IFN--induced effect against MV. The abrogation of the IFN- effect with L-tryptophan was not complete, suggesting that other IFN--induced mechanisms, in addition to that mediated by IDO, play a role. IL-1ß in combination with IFN- led to a synergistic induction of IDO. With IFN- alone, the maximal effect appeared to be exerted with 1,000 U/ml IFN-, whereas in the presence of IL-1ß similar effects were achieved already with 100 U/ml IFN-. The higher expression level of IDO, especially at lower IFN- concentrations (100 U/ml), was reflected by a stronger effect against MV in A549 cells. In HBMEC, this effect was less pronounced. To our knowledge, we describe here for the first time an IDO-mediated activity against a negative-strand RNA virus.

The enzyme IDO is induced in response to T-cell-produced IFN- and plays an important mechanistic role as part of the immune response, not only during infectious diseases but also in cancer and autoimmune diseases (8). The essential amino acid tryptophan is a source for the generation of serotonin and the formation of kynurenine derivatives and NAD. Of the two enzymes, tryptophan pyrrolase (tryptophan 2,3-dioxygenase) and IDO, involved in this process, tryptophan pyrrolase is located in liver cells, whereas the IFN--inducible enzyme IDO can be expressed by a wide range of nucleated cells. Because of the presence of IFN- at sites of inflammation, IDO is induced in a variety of infectious diseases, including viral infections such as human immunodeficiency virus, simian immunodeficiency virus, and human T-cell leukemia virus infections (9, 23, 32, 49). Its antiviral activity has recently been demonstrated for infections with CMV and HSV-1 (3, 7). The IFN--induced intracellular mechanisms against infectious agents such as viruses, bacteria, and parasites vary with respect to the cell type as well as the type of infectious agent. For example, inducible nitric oxide synthase (iNOS) is active against Toxoplasma gondii in murine macrophages, whereas in human macrophages an unknown mechanism different from that of IDO or iNOS is active. However, in the same cells, IDO is active against bacteria. Furthermore, IDO has been found to mediate antiparasitic activities in human fibroblasts, endothelial cells, and glioblastomas (1, 14, 31).

It was observed earlier that IFN- induces IDO only in certain primary cells or cell lines (57). In Table 1, we summarize the data concerning IDO induction and its antiviral activity. The capability of IFN- to induce IDO correlates in all cells tested so far with the induction of antiviral activity. IDO was readily induced in epithelial, endothelial, and astroglial cells, whereas neuronal cells and cells of the lymphoid line did not induce IDO in response to IFN-. Investigating the reason for the unresponsiveness of certain cells, we found with IMR-32 cells that STAT1 activation was delayed and reduced and that there was no induction of IDO at the level of mRNA (not shown), suggesting that IFN--receptor signaling is different in neuroblastoma cells. Monocytes and differentiated macrophages can react upon the induction of IDO to stimuli such as lipopolysaccharide via Toll-like receptors (22). Interestingly, the IFN-/ß-induced anti-MV activity mediated by MxA is also cell type specific, with a specific effect on the synthesis of viral glycoproteins in monocytes (52, 53). Dendritic cells expressing IDO suppress rather than stimulate the activity of contacting T cells (59). These findings demonstrate the importance of the cell type when antiviral effects of interferons are studied.

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TABLE 1. Correlation of IFN--induced IDO activity with antiviral activity in various cells

Among cells of neural origins, we detected IDO in glial cell lines but not in neuronal cell lines. In vivo, IDO expression was found in microglia, macrophages, astrocytes, and neurons, and in primary human neural cell cultures, IDO expression was also detected in astrocytes, microglia, and neurons (24, 28). It remains to be investigated in which human neurons IDO can be induced in vivo and whether it has a direct intracellular antiviral effect. With animal models, it has been demonstrated that IFN- can lead to a noncytolytic clearance of viruses from glial cells (45) and neurons (46). The clearance from neurons may depend on the neuronal type and the central nervous system region affected (6). Interestingly, IFN- is produced not only by activated T and NK cells but also by neurons in the brain (18, 40), and it exerts antiviral activity in certain neurons (17). However, IFN- also has negative effects in the nervous system since it interferes with myelination (4, 50), and transgenic mice expressing IFN- in the central nervous system have an overall decrease in myelination and a tremoring phenotype (13). Further experimental work is required to investigate the role of the seemingly contradictory antiviral and immunosuppressive effects of IDO and its metabolites on the pathogenesis of viral infections. Our findings that IFN- induces intracellular anti-MV activities mediated by IDO in epithelial, endothelial, and astroglial cells have revealed a further interesting aspect of the immune response against MV in various organs.

 
We thank the Deutsche Forschungsgemeinschaft for financial support (SPP 1130).

 
* Corresponding author. Mailing address: Institut für Virologie und Immunbiologie, Versbacher Str. 7, D-97078 Würzburg, Germany. Phone: 49-931-20149895. Fax: 49-931-20149553. E-mail: jss{at}vim.uni-wuerzburg.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, June 2005, p. 7768-7776, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7768-7776.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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