- Email: [email protected]
Impaired Microglial Activation in the Brain of IL-18-Gene-Disrupted Mice after Neurovirulent Influenza A Virus Infection
Virology 287, 163–170 (2001) doi:10.1006/viro.2001.1029, available online at http://www.idealibrary.com on
Impaired Microglial Activation in the Brain of IL-18-Gene-Disrupted Mice after Neurovirulent Influenza A Virus Infection Isamu Mori,* Md. Jaber Hossain,* Kiyoshi Takeda,† Haruki Okamura,‡ Yoshinori Imai,§ Shinichi Kohsaka,§ and Yoshinobu Kimura* ,1 *Department of Microbiology, Fukui Medical University School of Medicine, Fukui, Japan; †Department of Host Defenses, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; ‡Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; and §Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan Received April 2, 2001; returned to author for revision May 1, 2001; accepted May 30, 2001; published online July 23, 2001 Knockout of the inerleukin-18 (IL-18) gene predisposed mice to impaired clearance of neurovirulent influenza A virusinfected neurons from the brain. In wild-type mice, IL-18 molecule-producing microglia/macrophages emerged in virally attacked regions as early as day 3 after infection. Microglial transformation into macrophages culminated at day 7 to 9, with upregulated expression of Iba1, a novel calcium-binding protein that controls phagocytic functions of microglia/macrophages. In IL-18⫺/⫺ mice, microglial transformation was interrupted with reduced Iba1 expression. Interferon-␥ (IFN-␥)immunopositive neurons appeared in and around virally invaded regions in wild-type mice, peaking in number at day 7, whereas such cells were barely detected in IL-18⫺/⫺ mice. Stereotaxic microinjection of recombinant IFN-␥ triggered microglial transformation in IL-18⫺/⫺ mice and upregulated Iba1 expression, leading to effective eradication of virally infected neurons. Collectively, these results suggest that IL-18 plays a key role in activating microglial functions directed against the influenza virus infection by inducing neuronal IFN-␥ in the brain parenchyma. © 2001 Academic Press Key Words: interleukin-18; microglia; macrophages; Iba1; interferon-␥; influenza virus; neuron; brain; apoptosis; neurodegeneration.
INTRODUCTION
probably reflecting its developmental role in the central nervous system (CNS) (Prinz and Hanisch, 1999). Cultured microglial cells express IL-18 upon stimulation with lipopolysaccharide (Conti et al., 1999; Prinz and Hanisch, 1999). However, there has been no evidence of expression of IL-18 molecules in more mature brains, and it is still unclear whether IL-18 is expressed in the brain upon exposure to a certain insult, including viral infection of the CNS. Influenza viruses have been associated with a broad spectrum of CNS complications extending from acute encephalitis/encephalopathy to neuropsychiatric disorders (Mori and Kimura, 2001). Accumulating evidence has suggested the potential neuroinvasiveness of influenza viruses for humans (Frankova et al., 1977; Fujimoto et al., 1998; Gamboa et al., 1974; Hakoda and Nakatani, 2000; Murphy and Hawkes, 1970; Takahashi et al., 2000; Togashi et al., 2000). However, knowledge is scarce about the immune mechanism that exterminates the virus from the CNS as well as the immunopathology induced by the virus; hence, no effective treatment has been developed. Here we describe the early expression of IL-18 molecules in the mouse brain after neurovirulent influenza A virus infection and the defense mechanism conferred by this immunomudulatory cytokine, focusing especially
Interleukin-18 (IL-18), originally designated interferon-␥ (IFN-␥)-inducing factor, is a newly cloned pleiotropic cytokine that is synthesized mainly by activated macrophages (Okamura et al., 1998). IL-18 induces IFN-␥ production through Th1 cells, NK cells, and B cells (Kohno et al., 1997; Okamura et al., 1995b; Yoshimoto et al., 1997). IL-12 upregulates IL-18 receptor expression on T cells, Th1 cells, and B cells and brings about synergistic effects with IL-18 on IFN-␥ production (Sareneva et al., 2000; Yoshimoto et al., 1998). IL-18 and interferon-␣/ also synergize for IFN-␥ gene expression in T cells (Sareneva et al., 1998). IL-18 enhances Fas ligand-mediated cytotoxicity of Th1 and NK cells as well as perforin-mediated NK cell activity (Dao et al., 1996; Hyodo et al., 1999; Tsutsui et al., 1996). Thus, IL-18, in concert with other immunomodulatory molecules, may play a significant role in amplifying cytotoxic activities against unwanted cells, encompassing virally infected cells. Interestingly, it has been shown that IL-18 is expressed in the mouse brain during early postnatal stages,
1 To whom correspondence and reprint requests should be addressed at Department of Microbiology, Fukui Medical University School of Medicine, 23-3 Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan. Fax: 81-776-61-8104. E-mail: [email protected].
163
0042-6822/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
164
MORI ET AL.
Appearance of IL-18 molecules in the mouse brain after the WSN virus infection
FIG. 1. Kinetics in the number of WSN virus-immunolabeled neurons in the ipsilateral AON of wild-type (black bars) and IL-18⫺/⫺ mice (gray bars) after microinjection of the virus into the right olfactory bulb. Virus-infected cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each group for each time point). *Statistically significant (P ⬍ 0.01) by Student’s t test.
upon microglial actions that serve as the first line of defense against viral infections of the CNS. RESULTS Impaired elimination of the influenza WSN virusinfected neurons in the brain of IL-18⫺/⫺ mice The influenza A/WSN/33 virus, introduced into the right olfactory bulb of C57BL/6 mice, causes nonlethal acute encephalitis in mice with a high propensity to infect selectively restricted neurons in the brain parenchyma (Mori et al., 1999). This phenomenon is reproduced in this study. Thus, clusters of infected neurons appeared in the anterior olfactory nucleus (AON), piriform cortex, habenular and thalamic midline paraventricular nuclei, and hypothalamus. The time course study showed that WSN virus-immunopositive neurons emerged in the AON as early as day 1 after infection (Fig. 1). Thereafter, the infected neurons increased in number, peaking at day 5 in wild-type mice and at day 7 in IL-18⫺/⫺ mice. At day 7, the larger number of virally infected neurons in IL18⫺/⫺ mice than in wild-type mice was statistically significant (P ⬍ 0.01 by Student’s t test). The infected neurons were eliminated by day 14 in wild-type mice, whereas a small number of WSN virus-immunopositive degenerating neurons still remained in the brain of IL18⫺/⫺ mice. We have recently unraveled the process by which WSN virus-infected neurons die through apoptosis in the brain of C57BL/6 mice (Mori and Kimura, 2000). At day 7, the cresyl violet staining method disclosed that most neurons in the virally attacked areas of IL-18⫺/⫺ mice became shrunken, displaying peculiar nuclei (i.e., fragmented nuclei and condensed chromatin) as in the case of wild-type mice, suggesting that the infected neurons were dying through mechanisms associated with apoptosis (Mori and Kimura, 2000).
In virally attacked areas of the AON, IL-18-immunopositive cells emerged as early as day 3 after infection in wild-type mice (Fig. 2). A small fraction of cells immunopositive for IL-18 molecules displayed rod-shaped morphology (Kreutzberg, 1996). Most IL-18-positive cells, however, presented mixed morphological features of both microglia and macrophages, i.e., large cellular size (around 10 m in diameter) and round or irregular cellular outline with processes (Figs. 3A and 3B). To identify the IL-18-bearing cells, we immunostained brain tissues doubly for IL-18 and Iba1, a novel EF-hand Ca 2⫹-binding protein that is selectively expressed in cells belonging to the monocyte/macrophage lineage including microglia (Imai et al., 1996; Ito et al., 1998). The dual immunolabeling unequivocally demonstrated that the IL-18-bearing cells express Iba1 molecules, indicating that the cells were identical to microglia/macrophages (Fig. 3B). In other brain parenchymal regions where the virus infection did not reach, the immunohistochemistry did not detect any IL-18 molecule-expressing cells. The time course study showed that IL-18-bearing cells peaked in number at day 3 after infection, substantially decreased at day 5, and eventually disappeared by day 9 (Fig. 2). Microinjection of UV-inactivated virus into the right olfactory bulb did not induce IL-18-immunopositive cells in the mouse brain. IL-18-immunopositive cells did not emerge in the brain of IL-18⫺/⫺ mice after infection with the WSN virus at any time point tested. Transformation of microglial cells into macrophages In the previous study we found that resting microglia transform themselves into an activated state of rodshaped cells in response to influenza virus infection throughout the brain (Mori et al., 2000). Such activated
FIG. 2. Time course study of the number of IL-18 molecule-immunopositive cells that appeared in the ipsilateral AON of wild-type mice. IL-18 molecule-producing cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
IL-18 IN CONTROLLING CNS VIRUS INFECTION
165
FIG. 3. IL-18-bearing cells observed in the virally invaded area of the AON at day 3 after infection. (A) IL-18-immunolabeled cells (green) that displayed morphological features of microglia/macrophages. The bar marker represents 10 m. (B) Overlapped image of IL-18 and Iba1, a novel EF-hand calcium-binding protein that is expressed in microglia/macrophages. The IL-18-specific signal (green) was superimposed upon the Iba1-specific signal (red) to produce the mixed confocal image (yellow). The arrow indicates an IL-18 ⫹/Iba1 ⫹ cell that displayed mixed morphological features of microglia/macrophages. The arrowhead points to a small cell with IL-18 ⫺/Iba1 ⫹ phenotype, most likely representing a resting microglial cell. The bar marker represents 10 m.
microglia further transform themselves into macrophagelike cells (i.e., microglia-derived brain macrophages) especially in virus-infected areas of the brain where infected neurons are degenerating. In wild-type mice, several Iba-immunopositive macrophages appeared in the virally attacked regions of the AON at days 7 and 9 (Fig. 4). All those macrophages showed a strong Iba-1 immunoreactivity (Fig. 5A). In sharp contrast, Iba1-immunoreactive macrophages did not appear in the brain of IL18⫺/⫺ mice throughout the course of infection. Only rod-shaped or small round cells with faint Iba1 immunoreactivity emerged where infected neurons were present (Fig. 5B).
phology (Figs. 7A and 7B) and the dual immunolabeling for IFN-␥ and neuron-specific enolase demonstrated that these IFN-␥-producing cells were definitely neurons (Fig. 7E). IFN-␥ immunoreactivity was detected mainly in the
Induction of IFN-␥ molecules in the mouse brain Because IL-18 is a potent IFN-␥ inducer, we have investigated the expression of IFN-␥ molecules in the brain parenchyma after infection with the virus. In the brain of wild-type mice, cells with IFN-␥ immunoreactivity appeared as early as day 3 after infection, peaked in number at day 7, and eventually vanished by day 12 (Fig. 6). Such IFN-␥-bearing cells presented neuronal mor-
FIG. 4. Appearance of brain macrophages with Iba immunoreactivity in the virally attacked area of the ipsilateral AON in wild-type mice. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
FIG. 5. Microglial transformation into brain macrophages in response to WSN virus infection. (A) A cluster of brain macrophages with eminent Iba1 immunoreactivity found in the virally attacked area of the AON of wild-type mice at day 7 after infection (arrows). (B) Rod-shaped cells (arrowheads) intermixed with small round cells, displaying weak Iba1 immunoreactivity, observed at day 7 in the virus-infected region of the AON of IL-18⫺/⫺ mice. The bar marker in (B) represents 10 m and applies to (A) and (B).
166
MORI ET AL.
reduced the number of virally infected neurons in the ipsilateral AON at day 7 (IFN-␥ group, 11.7 ⫾ 0.3 for the AON area; PBS group, 102.7 ⫾ 17.5 for the AON area; P ⬍ 0.05 by Student’s t test; data are expressed as averages ⫾ SEM, n ⫽ 3 for each group) and eventually eradicated them by day 14. DISCUSSION
FIG. 6. A time course study of the occurrence of cells with IFN-␥ immunoreactivity in the ipsilateral AON of wild-type mice after infection with the WSN virus. IFN-␥-immunopositive cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
perikarya of neurons, but it was also observed in neurites on occasion. Such IFN-␥-producing neurons appeared abundantly in virus-attacked areas of the brain parenchyma (Fig. 7A), but they were also scattered in tissues such as the tenia tecta where viral infection did not reach (Fig. 7B). In sharp contrast to wild-type mice, no cells immunopositive for IFN-␥ occurred in the brain of IL-18⫺/⫺ mice (Fig. 7C), suggesting the impaired induction of IFN-␥ molecules in the CNS of these immunodefective mice. Induction of microglial response by administering the exogenous IFN-␥ into the brain of IL-18⫺/⫺ mice To confirm that impaired IFN-␥ expression in the brain of IL-18⫺/⫺ mice leads to interrupted microglial transformation into brain macrophages, we directly administered recombinant murine IFN-␥ into the AON and monitored the appearance of brain macrophages and the clearance of virally infected neurons. Microinjection of phosphate-buffered saline (PBS) into the AON did not influence the level of microglial activation (Fig. 8A). Microinjection of 1 or 10 units of IFN-␥ into the AON induced microglial transformation into macrophages with strong Iba1 immunoreactivity only in the vicinity of the needle track (Fig. 8B). Furthermore, introduction of 100 units of IFN-␥ into the AON induced brain macrophages that were widespread in the brain section ipsilaterally, including the AON and the frontal cortex (Fig. 8C). Upon staining with cresyl violet, no significant neurodegeneration was detected in the brain except the needle track injury. Stereotaxic introduction of recombinant murine IFN-␥ into the right AON at day 5 after infection induced Iba1-immunopositive brain macrophages at day 7 (IFN-␥ group, 11.0 ⫾ 2.0 for the AON area; PBS group, 0.3 ⫾ 0.3 for the AON area; P ⬍ 0.01 by Student’s t test; data are expressed as averages ⫾ SEM, n ⫽ 3 for each group)
IL-18 molecules appeared exclusively in microglia/ macrophages in the brain of C57BL/6 mice at the early time point of day 3 after infection. To our knowledge, this is the first report describing IL-18 expression in microglia/macrophages in response to infection of the CNS. These IL-18-producing microglia/macrophages emerged strictly in virus-infected regions of the brain parenchyma, alluding to this molecule’s possible role in amplifying the immune response. Expression of IL-18 was followed by the induction of neuronal IFN-␥ and successively by microglial transformation into macrophages with upregulated expression of Iba1 molecules. Knockout of the IL-18 gene led mice to a more widespread and prolonged WSN virus infection in the brain parenchyma in conjunction with impaired microglial activation. Extensive evidence has suggested that microglia play a central role in the first line of defense in the CNS (Kreutzberg, 1996). Microglia are rapidly activated in response to a certain insult given to the brain in a stereotyped and graded fashion (Kreutzberg, 1996). In the first stage (the nonphagocytic state), resident resting microglia become activated but do not become phagocytic. In the second stage (the phagocytic state), activated microglia further transform themselves into brain macrophages, if the insult to neurons is lethal. Intriguingly, microglial transformation into brain macrophages was interrupted in the brain of IL-18⫺/⫺ mice, which may give a reasonable explanation to delayed clearance of virally infected apoptotic neurons in these mice. To address the mechanism of IL-18-dependent microglial activation in the mouse brain, we have monitored IFN-␥ expression in the brain parenchyma after infection. Contrary to expectation, neurons, rather than lymphocytes, exhibited conspicuous IFN-␥ immunolabeling. Such IFN-␥ immunoreactivity might be identical to neuronal IFN-␥, which was originally purified from rat trigeminal ganglia by immunoaffinity chromatography and has been well characterized in vitro (Olsson et al., 1994). Its molecular weight is distinct from that of lymphocytederived IFN-␥, although these two molecules share some important biological functions such as control of cell proliferation, induction of the major histocompatibility complex class I and II, and antiviral effects. Neuronal IFN-␥ immunoreactivity is selectively expressed in the perikarya of neurons in the hypothalamic tuberomammillary nucleus and dorsal pontine tegmentum of the normal
IL-18 IN CONTROLLING CNS VIRUS INFECTION
rat brain (Bentivoglio et al., 1994). Neuronal IFN-␥ appears in the cytoplasm of axotomized motor neurons, and upregulated expression of neuronal IFN-␥ is recorded in response to infection with Sendai and mumps viruses in cultured sensory neurons (Eneroth et al., 1992; Olsson et al., 1989). Our results showed that no IFN-␥ immunoreactivity was detected in the brain of IL-18⫺/⫺ mice, which may explain the impaired microglial activation in these immunodefective mice. Direct administration of recombinant IFN-␥ into the brain of IL-18⫺/⫺ mice triggered microglial transformation, upregulation of Iba1 expression, and clearance of virally infected neurons, demonstrating the importance of IFN-␥ in inducing some important microglial functions directed against the viral infection. Recently we showed that Iba1 molecules are involved in membrane ruffling, and thus cell migration, and phagocytic functions of microglia/macrophages (Ohsawa et al., 2000). Biological actions of IFN-␥ in the CNS, mediated by inducible nitric oxide synthetase-derived nitric oxide, can be detrimental by expediting neuronal death leading to neuropathology, while they can be beneficial by promoting the differentiation and survival of neurons (MunozFernandez and Fresno, 1998). Accordingly, it appears reasonable to speculate that IFN-␥, produced in virusattacked areas, may facilitate cytotoxic and phagocytic activities in order to exterminate virally infected neurons, whereas that released in surrounding regions may provide neurons with direct antiviral actions and protective supports for survival. IL-18 production is downregulated on day 5 after infection and later, suggesting that IL-18 expression is strictly controlled in the brain. Prolonged and uncontrolled expression of IL-18 may give rise to intensified inflammation and tissue damage (Okamura et al., 1995a). On the other hand, IL-18 may offer a promising prophylactic and therapeutic strategy against intracellular microbes (Fujioka et al., 1999; Mastroeni et al., 1999; Ohkusu et al., 2000; Sugawara et al., 1999; Tanaka-Kataoka
167
et al., 1999). The pharmacological effects of this cytokine on influenza virus-induced encephalitis remain to be investigated in our experimental system. MATERIALS AND METHODS Experimental infection of animals Specific pathogen-free female C57BL/6J mice (Clea Japan, Osaka, Japan), 4 weeks of age, were used for experiments. IL-18⫺/⫺ mice, with a genetic background of C57BL/6 mice, were generated by the gene targeting method (Takeda et al., 1998) and were bred in The Animal Laboratory of Fukui Medical University. Female IL18⫺/⫺ mice were used for experiments at the age of 4 weeks. One microliter of the suspension containing 10 5 plaque-forming units of the influenza A/WSN/33 virus (a kind gift from Dr. S. Nakajima, The Institute of Public Health, Tokyo, Japan) was stereotaxically introduced into the right main olfactory bulb as described previously (Mori et al., 1999). UV inactivation of the virus Stock virus was inactivated with UV light as described previously (Mori et al., 1995). The titer of infectious influenza virus particles was reduced to ⬍10 ⫺7 of the original. Tissue processing Under deep anesthesia from the intraperitoneal administration of 7.2% chloral hydrate in PBS (0.05 ml/g body weight), mice were perfused with ice-cold 3.7% formaldehyde in PBS. The brains were dissected free of the cranium, soaked in 15% sucrose in PBS at 4°C overnight, and frozen at ⫺80°C. Coronal sections of 14-m thickness were cut on a cryostat at levels encompassing the AON and habenular/paraventricular nucleus, air-dried, and kept at ⫺30°C until staining.
FIG. 7. Neurons with IFN-␥ immunoreactivity that emerged in the brain of wild-type mice in response to the WSN virus infection. IFN-␥immunolabeled neurons observed (A) in the piriform cortex where a number of neurons became infected and (B) in the tenia tecta where viral infection did not reach. Seven days after infection. Note that neurites as well as cytoplasm of these neurons displayed IFN-␥ immunoreactivity. IFN-␥immunopositive neurons did not appear (C) in the AON of IL-18⫺/⫺ mice on day 7 after infection and (D) in the AON of wild-type mice after mock infection. The bar marker in (D) represents 10 m and applies to (A) through (D). (E) IFN-␥-producing cells with coexpression of neuron-specific enolase. The IFN-␥-specific signal (red) was superimposed upon neuron-specific signal (green) to produce the mixed confocal image (yellow). The arrows indicate IFN-␥-positive neurons in the virus-attacked region of the hypothalamus. Seven days after infection. The bar marker represents 10 m. FIG. 8. Induction of microglial transformation into brain macrophages by exogenous murine IFN-␥ in the brain of IL-18⫺/⫺ mice. (A) No microglial response was obtained by stereotaxically administering 1 l of PBS into the right AON. Resting microglia with faint Iba1 immunolabeling (arrowheads) were observed near the needle track injury (indicated by the asterisk). (B) Brain macrophages with strong Iba1 immunoreactivity (arrows) emerged only in the vicinity of the needle track injury (indicated by the asterisk) 48 h after microinjection of 1 or 10 units of recombinant murine IFN-␥ into the AON. (C) When mice were given 100 units of IFN-␥ in the right AON, Iba1-bearing brain macrophages (arrows) appeared 48 h after injection, widespread in the brain section ipsilaterally, including the AON, as shown in the figure, and the frontal cortex. (D) Microglial activation in the left side of the brain was not evident 48 h after introduction of 100 units of IFN-␥ into the right AON. Resting microglia weakly positive for Iba1 (arrowheads) were observed. The bar marker represents 10 m and applies to (A) through (D).
168
MORI ET AL.
IL-18 IN CONTROLLING CNS VIRUS INFECTION
In situ immunolabeling In situ single or dual immunolabeling of WSN virus antigens and various molecules in the brain slices was performed according to the protocol previously described (Mori et al., 1999, 2000) with the use of rabbit anti-WSN virus serum (a generous gift from Dr. S. Nakajima, The Institute for Public Health, Tokyo, Japan), rat monoclonal antibody against mouse IL-18 (clone 39-3F, IgG2ax; working concentration, 1.5 g/ml), affinity-purified rabbit anti-Iba-1 antibody (working concentration 2.0 g/ml), rat monoclonal antibody against murine IFN-␥ (PBL Biomedical Laboratories, New Brunswick, NJ; clone RMMG-1; working concentration, 6.25 g/ml), and mouse monoclonal antibody against neuron-specific enolase (Chemicon International, Temecula, CA; MAB324; working concentration, 9 g/ml). The intestinal tissue of a mouse was used as a positive control for IL-18 molecules (Takeuchi et al., 1997). Negative controls included the normal brain and sham-staining without the primary antibody. The immunolabeling was visualized under the Olympus GB200-UV-H-SET confocal laser scanning microscope (Olympus Optical Co. Ltd., Tokyo, Japan) at an excitation wavelength of 488 nm. An adjacent series of sections was stained with cresyl violet for histopathological and cytoarchitectonic observation as well as for detection of morphological alterations characteristic of apoptosis. Stereotaxic microinjection of recombinant murine IFN-␥ into the brain Mice were anesthetized with 7.2% chloral hydrate in PBS (0.005 ml/g body weight) and immobilized in a stereotaxic frame. The stereotaxic coordinates relative to the bregma were 2.6 mm rostral, 0.9 mm right lateral, and 4.0 mm ventral. A 1-l suspension of recombinant murine IFN-␥ (Life Technologies, Gaithersburg, MD), containing 0, 1, 10, or 100 units in PBS, was injected into the right AON with a Hamilton syringe at a rate of 0.5 l/min. The needle was retained in situ for a further 5 min after injection. REFERENCES Bentivoglio, M., Florenzano, F., Peng, Z. C., and Kristensson, K. (1994). Neuronal IFN-␥ in tuberomammillary neurones. NeuroReport 5, 2413–2416. Conti, B., Park, L. C., Calingasan, N. Y., Kim, Y., Kim, H., Bae, Y., Gibson, G. E., and Joh, T. H. (1999). Cultures of astrocytes and microglia express interleukin 18. Brain Res. Mol. Brain Res. 67, 46–52. Dao, T., Ohashi, K., Kayano, T., Kurimoto, M., and Okamura, H. (1996). Interferon-␥-inducing factor, a novel cytokine, enhances Fas ligandmediated cytotoxicity of murine T helper 1 cells. Cell. Immunol. 173, 230–235. Eneroth, A., Andersson, T., Olsson, T., Orvell, C., Norrby, E., and Kristensson, K. (1992). Interferon-␥-like immunoreactivity in sensory neurons may influence the replication of Sendai and mumps viruses. J. Neurosci. Res. 31, 487–493.
169
Frankova, V., Jirasek, A., and Tumova, B. (1977). Type A influenza: Postmortem virus isolations from different organs in human lethal cases. Arch. Virol. 53, 265–268. Fujimoto, S., Kobayashi, M., Uemura, O., Iwasa, M., Ando, T., Katoh, T., Nakamura, C., Maki, N., Togari, H., and Wada, Y. (1998). PCR on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis. Lancet 352, 873–875. Fujioka, N., Akazawa, R., Ohashi, K., Fujii, M., Ikeda, M., and Kurimoto, M. (1999). Interleukin-18 protects mice against acute herpes simplex virus type 1 infection. J. Virol. 73, 2401–2409. Gamboa, E. T., Wolf, A., Yahr, M. D., Harter, D. H., Duffy, P. E., Barden, H., and Hsu, K. C. (1974). Influenza virus antigen in postencephalitic parkinsonism brain. Detection by immunofluorescence. Arch. Neurol. 31, 228–232. Hakoda, S., and Nakatani, T. (2000). A pregnant woman with influenza A encephalopathy in whom influenza A/Hong Kong virus (H3) was isolated from cerebrospinal fluid. Arch. Intern. Med. 160, 1041, 1045. Hyodo, Y., Matsui, K., Hayashi, N., Tsutsui, H., Kashiwamura, S., Yamauchi, H., Hiroishi, K., Takeda, K., Tagawa, Y., Iwakura, Y., Kayagaki, N., Kurimoto, M., Okamura, H., Hada, T., Yagita, H., Akira, S., Nakanishi, K., and Higashino, K. (1999). IL-18 up-regulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor. J. Immunol. 162, 1662–1668. Imai, Y., Ibata, I., Ito, D., Ohsawa, K., and Kohsaka, S. (1996). A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem. Biophys. Res. Commun. 224, 855–862. Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y., and Kohsaka, S. (1998). Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res. Mol. Brain Res. 57, 1–9. Kohno, K., Kataoka, J., Ohtsuki, T., Suemoto, Y., Okamoto, I., Usui, M., Ikeda, M., and Kurimoto, M. (1997). IFN-␥-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158, 1541–1550. Kreutzberg, G. W. (1996). Microglia: A sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Mastroeni, P., Clare, S., Khan, S., Harrison, J. A., Hormaeche, C. E., Okamura, H., Kurimoto, M., and Dougan, G. (1999). Interleukin 18 contributes to host resistance and gamma interferon production in mice infected with virulent Salmonella typhimurium. Infect. Immun. 67, 478–483. Mori, I., Diehl, A. D., Chauhan, A., Ljunggren, H. G., and Kristensson, K. (1999). Selective targeting of habenular, thalamic midline and monoaminergic brainstem neurons by neurotropic influenza A virus in mice. J. Neurovirol. 5, 355–362. Mori, I., Imai, Y., Kohsaka, S., and Kimura, Y. (2000). Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection. Microbiol. Immunol. 44, 729–735. Mori, I., and Kimura, Y. (2000). Apoptotic neurodegeneration induced by influenza A virus infection in the mouse brain. Microbes Infect. 2, 1329–1334. Mori, I., and Kimura, Y. (2001). Neuropathogenesis of influenza virus infection in mice. Microbes Infect. 3, 475–479. Mori, I., Komatsu, T., Takeuchi, K., Nakakuki, K., Sudo, M., and Kimura, Y. (1995). Viremia induced by influenza virus. Microb. Pathog. 19, 237–244. Munoz-Fernandez, M. A., and Fresno, M. (1998). The role of tumour necrosis factor, interleukin 6, interferon-␥ and inducible nitric oxide synthase in the development and pathology of the nervous system. Prog. Neurobiol. 56, 307–340. Murphy, A. M., and Hawkes, R. A. (1970). Neurological complications of influenza A2-Hong Kong-68 virus. Med. J. Aust. 2, 511. Ohkusu, K., Yoshimoto, T., Takeda, K., Ogura, T., Kashiwamura, S.,
170
MORI ET AL.
Iwakura, Y., Akira, S., Okamura, H., and Nakanishi, K. (2000). Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection. Infect. Immun. 68, 2449–2456. Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y., and Kohsaka, S. (2000). Involvement of iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J. Cell Sci. 113(Pt. 17), 3073–3084. Okamura, H., Nagata, K., Komatsu, T., Tanimoto, T., Nukata, Y., Tanabe, F., Akita, K., Torigoe, K., Okura, T., Fukuda, S., et al. (1995a). A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. Infect. Immun. 63, 3966–3972. Okamura, H., Tsutsi, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., et al. (1995b). Cloning of a new cytokine that induces IFN-␥ production by T cells. Nature 378, 88–91. Okamura, H., Tsutsui, H., Kashiwamura, S., Yoshimoto, T., and Nakanishi, K. (1998). Interleukin-18: A novel cytokine that augments both innate and acquired immunity. Adv. Immunol. 70, 281–312. Olsson, T., Kelic, S., Edlund, C., Bakhiet, M., Hojeberg, B., van der Meide, P. H., Ljungdahl, A., and Kristensson, K. (1994). Neuronal interferon-␥ immunoreactive molecule: Bioactivities and purification. Eur. J. Immunol. 24, 308–314. Olsson, T., Kristensson, K., Ljungdahl, A., Maehlen, J., Holmdahl, R., and Klareskog, L. (1989). Gamma-interferon-like immunoreactivity in axotomized rat motor neurons. J. Neurosci. 9, 3870–3875. Prinz, M., and Hanisch, U. K. (1999). Murine microglial cells produce and respond to interleukin-18. J. Neurochem. 72, 2215–2218. Sareneva, T., Julkunen, I., and Matikainen, S. (2000). IFN-␥ and IL-12 induce IL-18 receptor gene expression in human NK and T cells. J. Immunol. 165, 1933–1938. Sareneva, T., Matikainen, S., Kurimoto, M., and Julkunen, I. (1998). Influenza A virus-induced IFN-␣/ and IL-18 synergistically enhance IFN-␥ gene expression in human T cells. J. Immunol. 160, 6032–6038. Sugawara, I., Yamada, H., Kaneko, H., Mizuno, S., Takeda, K., and Akira,
S. (1999). Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice. Infect. Immun. 67, 2585–2589. Takahashi, M., Yamada, T., Nakashita, Y., Saikusa, H., Deguchi, M., Kida, H., Tashiro, M., and Toyoda, T. (2000). Influenza virus-induced encephalopathy: Clinicopathologic study of an autopsied case. Pediatr. Int. 42, 204–214. Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K., and Akira, S. (1998). Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8, 383– 390. Takeuchi, M., Nishizaki, Y., Sano, O., Ohta, T., Ikeda, M., and Kurimoto, M. (1997). Immunohistochemical and immuno-electron-microscopic detection of interferon-␥-inducing factor (“interleukin-18”) in mouse intestinal epithelial cells. Cell Tissue Res. 289, 499–503. Tanaka-Kataoka, M., Kunikata, T., Takayama, S., Iwaki, K., Ohashi, K., Ikeda, M., and Kurimoto, M. (1999). In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection. Cytokine 11, 593–599. Togashi, T., Matsuzono, Y., and Narita, M. (2000). Epidemiology of influenza-associated encephalitis–encephalopathy in Hokkaido, the northernmost island of Japan. Pediatr. Int. 42, 192–196. Tsutsui, H., Nakanishi, K., Matsui, K., Higashino, K., Okamura, H., Miyazawa, Y., and Kaneda, K. (1996). IFN-␥-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157, 3967–3973. Yoshimoto, T., Okamura, H., Tagawa, Y. I., Iwakura, Y., and Nakanishi, K. (1997). Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-␥ production from activated B cells. Proc. Natl. Acad. Sci. USA 94, 3948–3953. Yoshimoto, T., Takeda, K., Tanaka, T., Ohkusu, K., Kashiwamura, S., Okamura, H., Akira, S., and Nakanishi, K. (1998). IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: Synergism with IL-18 for IFN-␥ production. J. Immunol. 161, 3400–3407.
Impaired Microglial Activation in the Brain of IL-18-Gene-Disrupted Mice after Neurovirulent Influenza A Virus Infection Isamu Mori,* Md. Jaber Hossain,* Kiyoshi Takeda,† Haruki Okamura,‡ Yoshinori Imai,§ Shinichi Kohsaka,§ and Yoshinobu Kimura* ,1 *Department of Microbiology, Fukui Medical University School of Medicine, Fukui, Japan; †Department of Host Defenses, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; ‡Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan; and §Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan Received April 2, 2001; returned to author for revision May 1, 2001; accepted May 30, 2001; published online July 23, 2001 Knockout of the inerleukin-18 (IL-18) gene predisposed mice to impaired clearance of neurovirulent influenza A virusinfected neurons from the brain. In wild-type mice, IL-18 molecule-producing microglia/macrophages emerged in virally attacked regions as early as day 3 after infection. Microglial transformation into macrophages culminated at day 7 to 9, with upregulated expression of Iba1, a novel calcium-binding protein that controls phagocytic functions of microglia/macrophages. In IL-18⫺/⫺ mice, microglial transformation was interrupted with reduced Iba1 expression. Interferon-␥ (IFN-␥)immunopositive neurons appeared in and around virally invaded regions in wild-type mice, peaking in number at day 7, whereas such cells were barely detected in IL-18⫺/⫺ mice. Stereotaxic microinjection of recombinant IFN-␥ triggered microglial transformation in IL-18⫺/⫺ mice and upregulated Iba1 expression, leading to effective eradication of virally infected neurons. Collectively, these results suggest that IL-18 plays a key role in activating microglial functions directed against the influenza virus infection by inducing neuronal IFN-␥ in the brain parenchyma. © 2001 Academic Press Key Words: interleukin-18; microglia; macrophages; Iba1; interferon-␥; influenza virus; neuron; brain; apoptosis; neurodegeneration.
INTRODUCTION
probably reflecting its developmental role in the central nervous system (CNS) (Prinz and Hanisch, 1999). Cultured microglial cells express IL-18 upon stimulation with lipopolysaccharide (Conti et al., 1999; Prinz and Hanisch, 1999). However, there has been no evidence of expression of IL-18 molecules in more mature brains, and it is still unclear whether IL-18 is expressed in the brain upon exposure to a certain insult, including viral infection of the CNS. Influenza viruses have been associated with a broad spectrum of CNS complications extending from acute encephalitis/encephalopathy to neuropsychiatric disorders (Mori and Kimura, 2001). Accumulating evidence has suggested the potential neuroinvasiveness of influenza viruses for humans (Frankova et al., 1977; Fujimoto et al., 1998; Gamboa et al., 1974; Hakoda and Nakatani, 2000; Murphy and Hawkes, 1970; Takahashi et al., 2000; Togashi et al., 2000). However, knowledge is scarce about the immune mechanism that exterminates the virus from the CNS as well as the immunopathology induced by the virus; hence, no effective treatment has been developed. Here we describe the early expression of IL-18 molecules in the mouse brain after neurovirulent influenza A virus infection and the defense mechanism conferred by this immunomudulatory cytokine, focusing especially
Interleukin-18 (IL-18), originally designated interferon-␥ (IFN-␥)-inducing factor, is a newly cloned pleiotropic cytokine that is synthesized mainly by activated macrophages (Okamura et al., 1998). IL-18 induces IFN-␥ production through Th1 cells, NK cells, and B cells (Kohno et al., 1997; Okamura et al., 1995b; Yoshimoto et al., 1997). IL-12 upregulates IL-18 receptor expression on T cells, Th1 cells, and B cells and brings about synergistic effects with IL-18 on IFN-␥ production (Sareneva et al., 2000; Yoshimoto et al., 1998). IL-18 and interferon-␣/ also synergize for IFN-␥ gene expression in T cells (Sareneva et al., 1998). IL-18 enhances Fas ligand-mediated cytotoxicity of Th1 and NK cells as well as perforin-mediated NK cell activity (Dao et al., 1996; Hyodo et al., 1999; Tsutsui et al., 1996). Thus, IL-18, in concert with other immunomodulatory molecules, may play a significant role in amplifying cytotoxic activities against unwanted cells, encompassing virally infected cells. Interestingly, it has been shown that IL-18 is expressed in the mouse brain during early postnatal stages,
1 To whom correspondence and reprint requests should be addressed at Department of Microbiology, Fukui Medical University School of Medicine, 23-3 Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan. Fax: 81-776-61-8104. E-mail: [email protected].
163
0042-6822/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
164
MORI ET AL.
Appearance of IL-18 molecules in the mouse brain after the WSN virus infection
FIG. 1. Kinetics in the number of WSN virus-immunolabeled neurons in the ipsilateral AON of wild-type (black bars) and IL-18⫺/⫺ mice (gray bars) after microinjection of the virus into the right olfactory bulb. Virus-infected cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each group for each time point). *Statistically significant (P ⬍ 0.01) by Student’s t test.
upon microglial actions that serve as the first line of defense against viral infections of the CNS. RESULTS Impaired elimination of the influenza WSN virusinfected neurons in the brain of IL-18⫺/⫺ mice The influenza A/WSN/33 virus, introduced into the right olfactory bulb of C57BL/6 mice, causes nonlethal acute encephalitis in mice with a high propensity to infect selectively restricted neurons in the brain parenchyma (Mori et al., 1999). This phenomenon is reproduced in this study. Thus, clusters of infected neurons appeared in the anterior olfactory nucleus (AON), piriform cortex, habenular and thalamic midline paraventricular nuclei, and hypothalamus. The time course study showed that WSN virus-immunopositive neurons emerged in the AON as early as day 1 after infection (Fig. 1). Thereafter, the infected neurons increased in number, peaking at day 5 in wild-type mice and at day 7 in IL-18⫺/⫺ mice. At day 7, the larger number of virally infected neurons in IL18⫺/⫺ mice than in wild-type mice was statistically significant (P ⬍ 0.01 by Student’s t test). The infected neurons were eliminated by day 14 in wild-type mice, whereas a small number of WSN virus-immunopositive degenerating neurons still remained in the brain of IL18⫺/⫺ mice. We have recently unraveled the process by which WSN virus-infected neurons die through apoptosis in the brain of C57BL/6 mice (Mori and Kimura, 2000). At day 7, the cresyl violet staining method disclosed that most neurons in the virally attacked areas of IL-18⫺/⫺ mice became shrunken, displaying peculiar nuclei (i.e., fragmented nuclei and condensed chromatin) as in the case of wild-type mice, suggesting that the infected neurons were dying through mechanisms associated with apoptosis (Mori and Kimura, 2000).
In virally attacked areas of the AON, IL-18-immunopositive cells emerged as early as day 3 after infection in wild-type mice (Fig. 2). A small fraction of cells immunopositive for IL-18 molecules displayed rod-shaped morphology (Kreutzberg, 1996). Most IL-18-positive cells, however, presented mixed morphological features of both microglia and macrophages, i.e., large cellular size (around 10 m in diameter) and round or irregular cellular outline with processes (Figs. 3A and 3B). To identify the IL-18-bearing cells, we immunostained brain tissues doubly for IL-18 and Iba1, a novel EF-hand Ca 2⫹-binding protein that is selectively expressed in cells belonging to the monocyte/macrophage lineage including microglia (Imai et al., 1996; Ito et al., 1998). The dual immunolabeling unequivocally demonstrated that the IL-18-bearing cells express Iba1 molecules, indicating that the cells were identical to microglia/macrophages (Fig. 3B). In other brain parenchymal regions where the virus infection did not reach, the immunohistochemistry did not detect any IL-18 molecule-expressing cells. The time course study showed that IL-18-bearing cells peaked in number at day 3 after infection, substantially decreased at day 5, and eventually disappeared by day 9 (Fig. 2). Microinjection of UV-inactivated virus into the right olfactory bulb did not induce IL-18-immunopositive cells in the mouse brain. IL-18-immunopositive cells did not emerge in the brain of IL-18⫺/⫺ mice after infection with the WSN virus at any time point tested. Transformation of microglial cells into macrophages In the previous study we found that resting microglia transform themselves into an activated state of rodshaped cells in response to influenza virus infection throughout the brain (Mori et al., 2000). Such activated
FIG. 2. Time course study of the number of IL-18 molecule-immunopositive cells that appeared in the ipsilateral AON of wild-type mice. IL-18 molecule-producing cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
IL-18 IN CONTROLLING CNS VIRUS INFECTION
165
FIG. 3. IL-18-bearing cells observed in the virally invaded area of the AON at day 3 after infection. (A) IL-18-immunolabeled cells (green) that displayed morphological features of microglia/macrophages. The bar marker represents 10 m. (B) Overlapped image of IL-18 and Iba1, a novel EF-hand calcium-binding protein that is expressed in microglia/macrophages. The IL-18-specific signal (green) was superimposed upon the Iba1-specific signal (red) to produce the mixed confocal image (yellow). The arrow indicates an IL-18 ⫹/Iba1 ⫹ cell that displayed mixed morphological features of microglia/macrophages. The arrowhead points to a small cell with IL-18 ⫺/Iba1 ⫹ phenotype, most likely representing a resting microglial cell. The bar marker represents 10 m.
microglia further transform themselves into macrophagelike cells (i.e., microglia-derived brain macrophages) especially in virus-infected areas of the brain where infected neurons are degenerating. In wild-type mice, several Iba-immunopositive macrophages appeared in the virally attacked regions of the AON at days 7 and 9 (Fig. 4). All those macrophages showed a strong Iba-1 immunoreactivity (Fig. 5A). In sharp contrast, Iba1-immunoreactive macrophages did not appear in the brain of IL18⫺/⫺ mice throughout the course of infection. Only rod-shaped or small round cells with faint Iba1 immunoreactivity emerged where infected neurons were present (Fig. 5B).
phology (Figs. 7A and 7B) and the dual immunolabeling for IFN-␥ and neuron-specific enolase demonstrated that these IFN-␥-producing cells were definitely neurons (Fig. 7E). IFN-␥ immunoreactivity was detected mainly in the
Induction of IFN-␥ molecules in the mouse brain Because IL-18 is a potent IFN-␥ inducer, we have investigated the expression of IFN-␥ molecules in the brain parenchyma after infection with the virus. In the brain of wild-type mice, cells with IFN-␥ immunoreactivity appeared as early as day 3 after infection, peaked in number at day 7, and eventually vanished by day 12 (Fig. 6). Such IFN-␥-bearing cells presented neuronal mor-
FIG. 4. Appearance of brain macrophages with Iba immunoreactivity in the virally attacked area of the ipsilateral AON in wild-type mice. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
FIG. 5. Microglial transformation into brain macrophages in response to WSN virus infection. (A) A cluster of brain macrophages with eminent Iba1 immunoreactivity found in the virally attacked area of the AON of wild-type mice at day 7 after infection (arrows). (B) Rod-shaped cells (arrowheads) intermixed with small round cells, displaying weak Iba1 immunoreactivity, observed at day 7 in the virus-infected region of the AON of IL-18⫺/⫺ mice. The bar marker in (B) represents 10 m and applies to (A) and (B).
166
MORI ET AL.
reduced the number of virally infected neurons in the ipsilateral AON at day 7 (IFN-␥ group, 11.7 ⫾ 0.3 for the AON area; PBS group, 102.7 ⫾ 17.5 for the AON area; P ⬍ 0.05 by Student’s t test; data are expressed as averages ⫾ SEM, n ⫽ 3 for each group) and eventually eradicated them by day 14. DISCUSSION
FIG. 6. A time course study of the occurrence of cells with IFN-␥ immunoreactivity in the ipsilateral AON of wild-type mice after infection with the WSN virus. IFN-␥-immunopositive cells were counted in the whole area of the section preparation. Data are expressed as averages ⫾ SEM (n ⫽ 3 for each time point).
perikarya of neurons, but it was also observed in neurites on occasion. Such IFN-␥-producing neurons appeared abundantly in virus-attacked areas of the brain parenchyma (Fig. 7A), but they were also scattered in tissues such as the tenia tecta where viral infection did not reach (Fig. 7B). In sharp contrast to wild-type mice, no cells immunopositive for IFN-␥ occurred in the brain of IL-18⫺/⫺ mice (Fig. 7C), suggesting the impaired induction of IFN-␥ molecules in the CNS of these immunodefective mice. Induction of microglial response by administering the exogenous IFN-␥ into the brain of IL-18⫺/⫺ mice To confirm that impaired IFN-␥ expression in the brain of IL-18⫺/⫺ mice leads to interrupted microglial transformation into brain macrophages, we directly administered recombinant murine IFN-␥ into the AON and monitored the appearance of brain macrophages and the clearance of virally infected neurons. Microinjection of phosphate-buffered saline (PBS) into the AON did not influence the level of microglial activation (Fig. 8A). Microinjection of 1 or 10 units of IFN-␥ into the AON induced microglial transformation into macrophages with strong Iba1 immunoreactivity only in the vicinity of the needle track (Fig. 8B). Furthermore, introduction of 100 units of IFN-␥ into the AON induced brain macrophages that were widespread in the brain section ipsilaterally, including the AON and the frontal cortex (Fig. 8C). Upon staining with cresyl violet, no significant neurodegeneration was detected in the brain except the needle track injury. Stereotaxic introduction of recombinant murine IFN-␥ into the right AON at day 5 after infection induced Iba1-immunopositive brain macrophages at day 7 (IFN-␥ group, 11.0 ⫾ 2.0 for the AON area; PBS group, 0.3 ⫾ 0.3 for the AON area; P ⬍ 0.01 by Student’s t test; data are expressed as averages ⫾ SEM, n ⫽ 3 for each group)
IL-18 molecules appeared exclusively in microglia/ macrophages in the brain of C57BL/6 mice at the early time point of day 3 after infection. To our knowledge, this is the first report describing IL-18 expression in microglia/macrophages in response to infection of the CNS. These IL-18-producing microglia/macrophages emerged strictly in virus-infected regions of the brain parenchyma, alluding to this molecule’s possible role in amplifying the immune response. Expression of IL-18 was followed by the induction of neuronal IFN-␥ and successively by microglial transformation into macrophages with upregulated expression of Iba1 molecules. Knockout of the IL-18 gene led mice to a more widespread and prolonged WSN virus infection in the brain parenchyma in conjunction with impaired microglial activation. Extensive evidence has suggested that microglia play a central role in the first line of defense in the CNS (Kreutzberg, 1996). Microglia are rapidly activated in response to a certain insult given to the brain in a stereotyped and graded fashion (Kreutzberg, 1996). In the first stage (the nonphagocytic state), resident resting microglia become activated but do not become phagocytic. In the second stage (the phagocytic state), activated microglia further transform themselves into brain macrophages, if the insult to neurons is lethal. Intriguingly, microglial transformation into brain macrophages was interrupted in the brain of IL-18⫺/⫺ mice, which may give a reasonable explanation to delayed clearance of virally infected apoptotic neurons in these mice. To address the mechanism of IL-18-dependent microglial activation in the mouse brain, we have monitored IFN-␥ expression in the brain parenchyma after infection. Contrary to expectation, neurons, rather than lymphocytes, exhibited conspicuous IFN-␥ immunolabeling. Such IFN-␥ immunoreactivity might be identical to neuronal IFN-␥, which was originally purified from rat trigeminal ganglia by immunoaffinity chromatography and has been well characterized in vitro (Olsson et al., 1994). Its molecular weight is distinct from that of lymphocytederived IFN-␥, although these two molecules share some important biological functions such as control of cell proliferation, induction of the major histocompatibility complex class I and II, and antiviral effects. Neuronal IFN-␥ immunoreactivity is selectively expressed in the perikarya of neurons in the hypothalamic tuberomammillary nucleus and dorsal pontine tegmentum of the normal
IL-18 IN CONTROLLING CNS VIRUS INFECTION
rat brain (Bentivoglio et al., 1994). Neuronal IFN-␥ appears in the cytoplasm of axotomized motor neurons, and upregulated expression of neuronal IFN-␥ is recorded in response to infection with Sendai and mumps viruses in cultured sensory neurons (Eneroth et al., 1992; Olsson et al., 1989). Our results showed that no IFN-␥ immunoreactivity was detected in the brain of IL-18⫺/⫺ mice, which may explain the impaired microglial activation in these immunodefective mice. Direct administration of recombinant IFN-␥ into the brain of IL-18⫺/⫺ mice triggered microglial transformation, upregulation of Iba1 expression, and clearance of virally infected neurons, demonstrating the importance of IFN-␥ in inducing some important microglial functions directed against the viral infection. Recently we showed that Iba1 molecules are involved in membrane ruffling, and thus cell migration, and phagocytic functions of microglia/macrophages (Ohsawa et al., 2000). Biological actions of IFN-␥ in the CNS, mediated by inducible nitric oxide synthetase-derived nitric oxide, can be detrimental by expediting neuronal death leading to neuropathology, while they can be beneficial by promoting the differentiation and survival of neurons (MunozFernandez and Fresno, 1998). Accordingly, it appears reasonable to speculate that IFN-␥, produced in virusattacked areas, may facilitate cytotoxic and phagocytic activities in order to exterminate virally infected neurons, whereas that released in surrounding regions may provide neurons with direct antiviral actions and protective supports for survival. IL-18 production is downregulated on day 5 after infection and later, suggesting that IL-18 expression is strictly controlled in the brain. Prolonged and uncontrolled expression of IL-18 may give rise to intensified inflammation and tissue damage (Okamura et al., 1995a). On the other hand, IL-18 may offer a promising prophylactic and therapeutic strategy against intracellular microbes (Fujioka et al., 1999; Mastroeni et al., 1999; Ohkusu et al., 2000; Sugawara et al., 1999; Tanaka-Kataoka
167
et al., 1999). The pharmacological effects of this cytokine on influenza virus-induced encephalitis remain to be investigated in our experimental system. MATERIALS AND METHODS Experimental infection of animals Specific pathogen-free female C57BL/6J mice (Clea Japan, Osaka, Japan), 4 weeks of age, were used for experiments. IL-18⫺/⫺ mice, with a genetic background of C57BL/6 mice, were generated by the gene targeting method (Takeda et al., 1998) and were bred in The Animal Laboratory of Fukui Medical University. Female IL18⫺/⫺ mice were used for experiments at the age of 4 weeks. One microliter of the suspension containing 10 5 plaque-forming units of the influenza A/WSN/33 virus (a kind gift from Dr. S. Nakajima, The Institute of Public Health, Tokyo, Japan) was stereotaxically introduced into the right main olfactory bulb as described previously (Mori et al., 1999). UV inactivation of the virus Stock virus was inactivated with UV light as described previously (Mori et al., 1995). The titer of infectious influenza virus particles was reduced to ⬍10 ⫺7 of the original. Tissue processing Under deep anesthesia from the intraperitoneal administration of 7.2% chloral hydrate in PBS (0.05 ml/g body weight), mice were perfused with ice-cold 3.7% formaldehyde in PBS. The brains were dissected free of the cranium, soaked in 15% sucrose in PBS at 4°C overnight, and frozen at ⫺80°C. Coronal sections of 14-m thickness were cut on a cryostat at levels encompassing the AON and habenular/paraventricular nucleus, air-dried, and kept at ⫺30°C until staining.
FIG. 7. Neurons with IFN-␥ immunoreactivity that emerged in the brain of wild-type mice in response to the WSN virus infection. IFN-␥immunolabeled neurons observed (A) in the piriform cortex where a number of neurons became infected and (B) in the tenia tecta where viral infection did not reach. Seven days after infection. Note that neurites as well as cytoplasm of these neurons displayed IFN-␥ immunoreactivity. IFN-␥immunopositive neurons did not appear (C) in the AON of IL-18⫺/⫺ mice on day 7 after infection and (D) in the AON of wild-type mice after mock infection. The bar marker in (D) represents 10 m and applies to (A) through (D). (E) IFN-␥-producing cells with coexpression of neuron-specific enolase. The IFN-␥-specific signal (red) was superimposed upon neuron-specific signal (green) to produce the mixed confocal image (yellow). The arrows indicate IFN-␥-positive neurons in the virus-attacked region of the hypothalamus. Seven days after infection. The bar marker represents 10 m. FIG. 8. Induction of microglial transformation into brain macrophages by exogenous murine IFN-␥ in the brain of IL-18⫺/⫺ mice. (A) No microglial response was obtained by stereotaxically administering 1 l of PBS into the right AON. Resting microglia with faint Iba1 immunolabeling (arrowheads) were observed near the needle track injury (indicated by the asterisk). (B) Brain macrophages with strong Iba1 immunoreactivity (arrows) emerged only in the vicinity of the needle track injury (indicated by the asterisk) 48 h after microinjection of 1 or 10 units of recombinant murine IFN-␥ into the AON. (C) When mice were given 100 units of IFN-␥ in the right AON, Iba1-bearing brain macrophages (arrows) appeared 48 h after injection, widespread in the brain section ipsilaterally, including the AON, as shown in the figure, and the frontal cortex. (D) Microglial activation in the left side of the brain was not evident 48 h after introduction of 100 units of IFN-␥ into the right AON. Resting microglia weakly positive for Iba1 (arrowheads) were observed. The bar marker represents 10 m and applies to (A) through (D).
168
MORI ET AL.
IL-18 IN CONTROLLING CNS VIRUS INFECTION
In situ immunolabeling In situ single or dual immunolabeling of WSN virus antigens and various molecules in the brain slices was performed according to the protocol previously described (Mori et al., 1999, 2000) with the use of rabbit anti-WSN virus serum (a generous gift from Dr. S. Nakajima, The Institute for Public Health, Tokyo, Japan), rat monoclonal antibody against mouse IL-18 (clone 39-3F, IgG2ax; working concentration, 1.5 g/ml), affinity-purified rabbit anti-Iba-1 antibody (working concentration 2.0 g/ml), rat monoclonal antibody against murine IFN-␥ (PBL Biomedical Laboratories, New Brunswick, NJ; clone RMMG-1; working concentration, 6.25 g/ml), and mouse monoclonal antibody against neuron-specific enolase (Chemicon International, Temecula, CA; MAB324; working concentration, 9 g/ml). The intestinal tissue of a mouse was used as a positive control for IL-18 molecules (Takeuchi et al., 1997). Negative controls included the normal brain and sham-staining without the primary antibody. The immunolabeling was visualized under the Olympus GB200-UV-H-SET confocal laser scanning microscope (Olympus Optical Co. Ltd., Tokyo, Japan) at an excitation wavelength of 488 nm. An adjacent series of sections was stained with cresyl violet for histopathological and cytoarchitectonic observation as well as for detection of morphological alterations characteristic of apoptosis. Stereotaxic microinjection of recombinant murine IFN-␥ into the brain Mice were anesthetized with 7.2% chloral hydrate in PBS (0.005 ml/g body weight) and immobilized in a stereotaxic frame. The stereotaxic coordinates relative to the bregma were 2.6 mm rostral, 0.9 mm right lateral, and 4.0 mm ventral. A 1-l suspension of recombinant murine IFN-␥ (Life Technologies, Gaithersburg, MD), containing 0, 1, 10, or 100 units in PBS, was injected into the right AON with a Hamilton syringe at a rate of 0.5 l/min. The needle was retained in situ for a further 5 min after injection. REFERENCES Bentivoglio, M., Florenzano, F., Peng, Z. C., and Kristensson, K. (1994). Neuronal IFN-␥ in tuberomammillary neurones. NeuroReport 5, 2413–2416. Conti, B., Park, L. C., Calingasan, N. Y., Kim, Y., Kim, H., Bae, Y., Gibson, G. E., and Joh, T. H. (1999). Cultures of astrocytes and microglia express interleukin 18. Brain Res. Mol. Brain Res. 67, 46–52. Dao, T., Ohashi, K., Kayano, T., Kurimoto, M., and Okamura, H. (1996). Interferon-␥-inducing factor, a novel cytokine, enhances Fas ligandmediated cytotoxicity of murine T helper 1 cells. Cell. Immunol. 173, 230–235. Eneroth, A., Andersson, T., Olsson, T., Orvell, C., Norrby, E., and Kristensson, K. (1992). Interferon-␥-like immunoreactivity in sensory neurons may influence the replication of Sendai and mumps viruses. J. Neurosci. Res. 31, 487–493.
169
Frankova, V., Jirasek, A., and Tumova, B. (1977). Type A influenza: Postmortem virus isolations from different organs in human lethal cases. Arch. Virol. 53, 265–268. Fujimoto, S., Kobayashi, M., Uemura, O., Iwasa, M., Ando, T., Katoh, T., Nakamura, C., Maki, N., Togari, H., and Wada, Y. (1998). PCR on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis. Lancet 352, 873–875. Fujioka, N., Akazawa, R., Ohashi, K., Fujii, M., Ikeda, M., and Kurimoto, M. (1999). Interleukin-18 protects mice against acute herpes simplex virus type 1 infection. J. Virol. 73, 2401–2409. Gamboa, E. T., Wolf, A., Yahr, M. D., Harter, D. H., Duffy, P. E., Barden, H., and Hsu, K. C. (1974). Influenza virus antigen in postencephalitic parkinsonism brain. Detection by immunofluorescence. Arch. Neurol. 31, 228–232. Hakoda, S., and Nakatani, T. (2000). A pregnant woman with influenza A encephalopathy in whom influenza A/Hong Kong virus (H3) was isolated from cerebrospinal fluid. Arch. Intern. Med. 160, 1041, 1045. Hyodo, Y., Matsui, K., Hayashi, N., Tsutsui, H., Kashiwamura, S., Yamauchi, H., Hiroishi, K., Takeda, K., Tagawa, Y., Iwakura, Y., Kayagaki, N., Kurimoto, M., Okamura, H., Hada, T., Yagita, H., Akira, S., Nakanishi, K., and Higashino, K. (1999). IL-18 up-regulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor. J. Immunol. 162, 1662–1668. Imai, Y., Ibata, I., Ito, D., Ohsawa, K., and Kohsaka, S. (1996). A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem. Biophys. Res. Commun. 224, 855–862. Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y., and Kohsaka, S. (1998). Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res. Mol. Brain Res. 57, 1–9. Kohno, K., Kataoka, J., Ohtsuki, T., Suemoto, Y., Okamoto, I., Usui, M., Ikeda, M., and Kurimoto, M. (1997). IFN-␥-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158, 1541–1550. Kreutzberg, G. W. (1996). Microglia: A sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Mastroeni, P., Clare, S., Khan, S., Harrison, J. A., Hormaeche, C. E., Okamura, H., Kurimoto, M., and Dougan, G. (1999). Interleukin 18 contributes to host resistance and gamma interferon production in mice infected with virulent Salmonella typhimurium. Infect. Immun. 67, 478–483. Mori, I., Diehl, A. D., Chauhan, A., Ljunggren, H. G., and Kristensson, K. (1999). Selective targeting of habenular, thalamic midline and monoaminergic brainstem neurons by neurotropic influenza A virus in mice. J. Neurovirol. 5, 355–362. Mori, I., Imai, Y., Kohsaka, S., and Kimura, Y. (2000). Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection. Microbiol. Immunol. 44, 729–735. Mori, I., and Kimura, Y. (2000). Apoptotic neurodegeneration induced by influenza A virus infection in the mouse brain. Microbes Infect. 2, 1329–1334. Mori, I., and Kimura, Y. (2001). Neuropathogenesis of influenza virus infection in mice. Microbes Infect. 3, 475–479. Mori, I., Komatsu, T., Takeuchi, K., Nakakuki, K., Sudo, M., and Kimura, Y. (1995). Viremia induced by influenza virus. Microb. Pathog. 19, 237–244. Munoz-Fernandez, M. A., and Fresno, M. (1998). The role of tumour necrosis factor, interleukin 6, interferon-␥ and inducible nitric oxide synthase in the development and pathology of the nervous system. Prog. Neurobiol. 56, 307–340. Murphy, A. M., and Hawkes, R. A. (1970). Neurological complications of influenza A2-Hong Kong-68 virus. Med. J. Aust. 2, 511. Ohkusu, K., Yoshimoto, T., Takeda, K., Ogura, T., Kashiwamura, S.,
170
MORI ET AL.
Iwakura, Y., Akira, S., Okamura, H., and Nakanishi, K. (2000). Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection. Infect. Immun. 68, 2449–2456. Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y., and Kohsaka, S. (2000). Involvement of iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J. Cell Sci. 113(Pt. 17), 3073–3084. Okamura, H., Nagata, K., Komatsu, T., Tanimoto, T., Nukata, Y., Tanabe, F., Akita, K., Torigoe, K., Okura, T., Fukuda, S., et al. (1995a). A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. Infect. Immun. 63, 3966–3972. Okamura, H., Tsutsi, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., et al. (1995b). Cloning of a new cytokine that induces IFN-␥ production by T cells. Nature 378, 88–91. Okamura, H., Tsutsui, H., Kashiwamura, S., Yoshimoto, T., and Nakanishi, K. (1998). Interleukin-18: A novel cytokine that augments both innate and acquired immunity. Adv. Immunol. 70, 281–312. Olsson, T., Kelic, S., Edlund, C., Bakhiet, M., Hojeberg, B., van der Meide, P. H., Ljungdahl, A., and Kristensson, K. (1994). Neuronal interferon-␥ immunoreactive molecule: Bioactivities and purification. Eur. J. Immunol. 24, 308–314. Olsson, T., Kristensson, K., Ljungdahl, A., Maehlen, J., Holmdahl, R., and Klareskog, L. (1989). Gamma-interferon-like immunoreactivity in axotomized rat motor neurons. J. Neurosci. 9, 3870–3875. Prinz, M., and Hanisch, U. K. (1999). Murine microglial cells produce and respond to interleukin-18. J. Neurochem. 72, 2215–2218. Sareneva, T., Julkunen, I., and Matikainen, S. (2000). IFN-␥ and IL-12 induce IL-18 receptor gene expression in human NK and T cells. J. Immunol. 165, 1933–1938. Sareneva, T., Matikainen, S., Kurimoto, M., and Julkunen, I. (1998). Influenza A virus-induced IFN-␣/ and IL-18 synergistically enhance IFN-␥ gene expression in human T cells. J. Immunol. 160, 6032–6038. Sugawara, I., Yamada, H., Kaneko, H., Mizuno, S., Takeda, K., and Akira,
S. (1999). Role of interleukin-18 (IL-18) in mycobacterial infection in IL-18-gene-disrupted mice. Infect. Immun. 67, 2585–2589. Takahashi, M., Yamada, T., Nakashita, Y., Saikusa, H., Deguchi, M., Kida, H., Tashiro, M., and Toyoda, T. (2000). Influenza virus-induced encephalopathy: Clinicopathologic study of an autopsied case. Pediatr. Int. 42, 204–214. Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K., and Akira, S. (1998). Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8, 383– 390. Takeuchi, M., Nishizaki, Y., Sano, O., Ohta, T., Ikeda, M., and Kurimoto, M. (1997). Immunohistochemical and immuno-electron-microscopic detection of interferon-␥-inducing factor (“interleukin-18”) in mouse intestinal epithelial cells. Cell Tissue Res. 289, 499–503. Tanaka-Kataoka, M., Kunikata, T., Takayama, S., Iwaki, K., Ohashi, K., Ikeda, M., and Kurimoto, M. (1999). In vivo antiviral effect of interleukin 18 in a mouse model of vaccinia virus infection. Cytokine 11, 593–599. Togashi, T., Matsuzono, Y., and Narita, M. (2000). Epidemiology of influenza-associated encephalitis–encephalopathy in Hokkaido, the northernmost island of Japan. Pediatr. Int. 42, 192–196. Tsutsui, H., Nakanishi, K., Matsui, K., Higashino, K., Okamura, H., Miyazawa, Y., and Kaneda, K. (1996). IFN-␥-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J. Immunol. 157, 3967–3973. Yoshimoto, T., Okamura, H., Tagawa, Y. I., Iwakura, Y., and Nakanishi, K. (1997). Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon-␥ production from activated B cells. Proc. Natl. Acad. Sci. USA 94, 3948–3953. Yoshimoto, T., Takeda, K., Tanaka, T., Ohkusu, K., Kashiwamura, S., Okamura, H., Akira, S., and Nakanishi, K. (1998). IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: Synergism with IL-18 for IFN-␥ production. J. Immunol. 161, 3400–3407.