Department of Microbiology, Fukui Medical University School of Medicine, 23-3 Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan1
Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi, Japan2
Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Aichi, Japan3
Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan4
Author for correspondence: Yoshinobu Kimura. Fax +81 776 61 8104. e-mail ykimura{at}fmsrsa.fukui-med.ac.jp
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Abstract |
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Introduction |
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Neurovirulent influenza A virus infection induces neuronal apoptosis in the mouse CNS following direct introduction of the virus into the olfactory bulb, depending on T cell-mediated mechanisms, including the perforin/granzyme and Fas/Fas L systems (Mori et al., 1999 ; Mori & Kimura, 2000
, 2001
).
Here we present evidence that ORNs die through apoptosis following intranasal infection of the recombinant R404BP strain of neurovirulent influenza A virus with upregulated expression of the Fas L molecules and activation of the c-Jun N-terminal kinase (JNK) cascade. The virus grown in ORNs did not invade the CNS or kill animals. The R404BP virus carries the matrix and neuraminidase genes of the neurovirulent WSN strain (H1N1) and other genes of the non-neurovirulent A/Aichi/2/68 strain (H3N2), showing an increased neurovirulent phenotype compared with the parental WSN virus. The virus, when introduced directly into the mouse CNS, spreads widely in the brain and kills 100% of the mice (Takahashi et al., 1995 ), suggesting a highly neurovirulent potential. We further show dynamic reactions of microglial cells in the olfactory epithelium, partaking in phagocytic activities for eventual clearing of apoptotic bodies.
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Methods |
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UV-inactivation of the virus.
Stock virus suspension was exposed to a 15 W UV lamp at a distance of 30 cm for 30 min at 4 °C with continuous and gentle stirring. After irradiation, infectivity was reduced to <10-7 of the original.
Tissue processing.
Under deep anaesthesia by the intraperitoneal administration of 7·2% chloral hydrate in PBS (0·05 ml/g body weight), mice were transcardially perfused with 3·7% formaldehyde in PBS. The nose was decalcified in 4 % ethylenediaminetetraacetic acid at room temperature for 7 days. The nose and brain were soaked in 20% sucrose in PBS at 4 °C overnight and frozen at -80 °C. Coronal sections of 14 µm thickness of the nose and brain tissues were cut on a cryostat.
Immunohistochemistry.
Immunohistochemistry was performed as described previously (Mori & Kimura, 2000 ). Primary antibodies included rabbit polyclonal anti-WSN virus antibody (a generous gift from Dr S. Nakajima), rabbit anti-inducible nitric oxide synthase (iNOS) antibody (working concentration of 5 µg/ml; Upstate Biotechnology), anti-nitrotyrosine (NT) antibody (working concentration of 20 µg/ml; Upstate Biotechnology) and rabbit polyclonal anti-Iba1 antibody (Imai et al., 1996
). The Iba1 molecules are expressed selectively in microglia/macrophages (Mori et al., 2000
).
Dual immunofluorescent labelling.
Tissue slices were incubated in 5% donkey serum (Chemicon International) containing 0·3% Triton-X in PBS for 20 min and reacted in a mixture of two primary antibodies diluted in 2% donkey serum containing 0·3% Triton-X in PBS at 4 °C overnight. The primary antibodies used were goat polyclonal anti-influenza A virus antibody (working dilution of 1:400; Chemicon International), rabbit polyclonal anti-neural cell adhesion molecule (NCAM) antibody (working concentration of 2·0 µg/ml; Chemicon International; Calof et al., 1996 ), rabbit polyclonal anti-cleaved caspase-3 antibody (working dilution of 1:50; Cell Signalling Technology), rabbit polyclonal anti-Fas antibody (working dilution of 1:400; Wako Pure Chemical Industries), rabbit polyclonal anti-Fas L antibody (working dilution of 1:200; Wako Pure Chemical Industries), rabbit polyclonal anti-Iba1 antibody (working concentration of 2·0 µg/ml), goat polyclonal anti-Fas L antibody (working dilution of 1:100; Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-JNK antibody (Thr183/Tyr185; working dilution of 1:200; Cell Signalling Technology) and anti-phospho-c-Jun antibody (Ser63; working dilution of 1:200; Cell Signalling Technology). Then, tissue sections were incubated in secondary antibodies affinity-purified and absorbed for dual immunolabelling (all diluted 1:100 in PBS supplemented with 0·3% Triton-X; Chemicon International), including fluorescein-labelled donkey anti-rabbit immunoglobulin, rodamine-labelled donkey anti-goat immunoglobulin, fluoroscein-labelled donkey anti-goat immunoglobulin and rodamine-labelled donkey anti-rabbit immunoglobulin. Binding was visualized under a confocal laser scanning microscope.
In situ detection of DNA fragmentation.
DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method, using an ApopTag Direct In Situ Apoptosis Detection kit (Intergen). Dual imaging for virus antigens and TUNEL reaction was carried out as described in a previous report (Mori & Kimura, 2000 ).
Detection of influenza virus matrix protein 1 (M1) mRNA.
Viral mRNA encoding influenza virus M1 was extracted from the nasal tissue and olfactory bulb by using an mRNA Isolation kit (Loche). cDNA synthesis and PCR were carried out with the One-Tube RTPCR system (Takara), according to the manufacturers instructions, with the sense primer 5' GAGATCGCACAGAGA 3' and the antisense primer 5' TCGTTGCATCTGCAC 3' (Urabe et al., 1993 ). The programme conditions were as follows: 50 °C for 30 min and 94 °C for 2 min, followed by 30 cycles of 94 °C for 15 s, 50 °C for 30 s and 72 °C for 45 s. The final products were differentiated on 1% agarose gel, stained with ethidium bromide and visualized under a UV lamp. The expected size of the product was 684 bp.
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Results |
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Infection of ORNs
The immunohistochemical study showed that infected cells appeared in the olfactory epithelium as early as day 1 after intranasal inoculation with the R404BP virus (Fig. 1c). Dual staining for viral antigens and the NCAM antigen, a marker of ORNs, proved that infected cells were identical primarily to the ORN (Fig. 2
). Clusters of infected ORNs and a small number of sustentacular cells, a kind of supporting cells in the olfactory epithelium, became infected on day 3 after infection (Fig. 1e
). On day 5, virally infected neurons began to shrink and split into small bodies (Fig. 1g
). On day 7, a virus antigen-immunopositive substance composed of small bodies appeared (Fig. 1i
), which vanished by day 14 (data not shown). The virus infection also occurred restrictedly in some ciliated respiratory epithelial cells facing the nasal cavity but did not reach the lung. RTPCR detected viral M1 mRNA in the nose from days 1 to 5 and proved negative on day 7 (Fig. 3
). Lymphocytic infiltration was not obvious in the neuroepithelium (Fig. 4
). The immunohistochemistry detected no iNOS or NT expression, indicators of inflammatory response, in virally attacked areas in the olfactory epithelium.
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The response of Iba1-immunopositive cells in the olfactory epithelium
Immunohistochemistry for Iba1 molecules, a specific marker of a monocytic-macrophage lineage, was performed in the consecutive nasal tissue slices. In the normal olfactory epithelium, small round cells with thick processes were observed (Fig. 1b). On day 1 after infection, rod-shaped cells that were immunopositive for Iba1 appeared in the corresponding area where infected ORNs were observed (Fig. 1d
). On day 3, cells presenting strong Iba1-immunoreactivity with thick processes were found (Fig. 1f
). On day 5, macrophage-like cells with prominent Iba1-immunoreactivity were detected (Fig. 1h
). On day 7, phagocyte-like cells (around 15 µm in diameter) with Iba1-immunoreactivity appeared in the corresponding regions where the virus antigen-immunopositive substance composed of small bodies emerged (Fig. 1j
). Such microglial activation did not take place in mice intranasally infected with UV-inactivated virus.
Demonstration of apoptosis in infected ORNs
Shrinkage of infected ORNs and appearance of small bodies were highly indicative of the occurrence of classical apoptosis. Cytoplasmic accumulation of cleaved caspase-3 in infected ORNs verified the occurrence of apoptosis (Fig. 4). A small number of uninfected cells with immunoreactivity for cleaved caspase-3 was also detected in areas surrounding a cluster of infected ORNs. The TUNEL-specific signal appeared at first in a nuclear staining pattern of infected ORNs on day 3 after infection (data not shown).
Activation of the Fas/Fas L system and JNK signal transduction pathway
In the normal olfactory neuroepithelium, Fas molecules were detected through the olfactory layer in a cytoplasmic staining fashion. Occasionally, the cell surface showed stronger Fas-specific signals typically as a dotted pattern (Fig. 5a). In sharp contrast, Fas L molecules were barely detectable through the layer of the olfactory epithelium (Fig. 5b
).
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Since the stress-activated signalling pathway appeared to be highly involved in the process of neuronal apoptosis (Putcha et al., 2001 ; Whitfield et al., 2001
), we investigated the expression of JNK (phosphorylated at Thr183 and Tyr185) and c-Jun (phosphorylated at Ser63) in infected ORNs. The immunofluorescence assay detected phosphorylated JNK and c-Jun in infected ORNs firstly on day 3, suggesting that the JNK/c-Jun signalling pathways become activated during the process of apoptosis induced by the R404BP virus infection (Fig. 6
).
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Discussion |
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Recent studies underscore the importance of the Fas/Fas L system in influenza virus-induced apoptosis in cultured cells. Influenza A virus infection upregulates the expression of Fas and Fas L molecules on the surface of HeLa cells, leading to apoptosis when infected cells come into contact with each other (Fujimoto et al., 1998 ). Furthermore, the Fas/Fas L signalling pathway has also been implicated in the occurrence of human lymphocyte apoptosis after influenza A virus exposure (Nichols et al., 2001
). Once upregulated, Fas L activates the apoptosis cascade in an autocrine or paracrine fashion by stimulating its receptor, Fas (Morishima et al., 2001
). We have shown that the neurovirulent influenza A virus induces Fas L in infected ORNs as well as sustentacular cells. Thus, it is possible that these infected cells and, at least in part, uninfected neighbouring cells, which are densely in contact with each other, undergo apoptosis through activation of the Fas/Fas L system. The JNK/c-Jun signal transduction pathway plays a critical role in the process of apoptosis of the nervous system, such as in cerebellar granule neurons, sympathetic neurons after withdrawal of nerve growth factor, hippocampal neurons in response to the excitotoxin kainic acid and cortical neurons exposed to
-amyloid (Morishima et al., 2001
; Whitfield et al., 2001). Activation of the JNK/c-Jun increases expression of the BH3-only protein BIM, which promotes BAX-dependent cytochrome c release from the mitochondria to the cytoplasm, leading to the formation of apoptosome (Putcha et al., 2001
; Whitfield et al., 2001
). On the other hand, activated JNK phosphorylates c-Jun, which stimulates transcription of several key molecules including Fas L. The JNK/c-JunFas/Fas L signalling pathway is highly implicated in dopamine- and
-amyloid-induced apoptosis in cellular models of Parkinsons and Alzheimers diseases, respectively (Luo et al., 1998
; Morishima et al., 2001
). In our experimental system, the JNK cascade also becomes activated in infected ORNs, suggesting the importance of this signal transduction system in the induction of neuronal apoptosis, which appears to be mediated by both the intrinsic (i.e. apoptosome) and the extrinsic (i.e. death receptor) pathways (Putcha et al., 2001
). Another mechanism might also be involved in the process of apoptosis of ORNs induced by the R404BP virus, which remains open for further studies. In cultured cortical neurons,
-amyloid activates calpain I, which cleaves p35, the regulatory subunit of cyclin-dependent kinase 5 (cdk5), to p25, leading to constitutive activation of cdk5 and, ultimately, neuronal apoptosis (Lee et al., 2000
).
Iba1 is an EF hand calcium-binding protein, specifically expressed in a monocytic cell line, including microglia (Imai et al., 1996 ; Ito et al., 1998
; Mori et al., 2000
). Iba1 has been proven to be an actin cross-linking protein involved in membrane ruffling and phagocytosis of macrophages/microglia (Ohsawa et al., 2000
; Sasaki et al., 2001
). We have found dynamic reactions of Iba1-expressing microglia-like cells in the olfactory epithelium in response to infection of ORNs with the neurovirulent influenza A virus. Microglial activation in the CNS takes place in a stereotypic and graded fashion irrespective of the cause of the lesion (Kreutzberg, 1996
; Mori et al., 2000
; Mori & Kimura, 2001
). In the first stage, when neuronal injury is sublethal, resting microglia become activated and produce trophic factors, contributing to tissue repair. In the second stage, when neuronal injury is lethal, activated microglia further transform themselves into phagocytic cells, known as microglia-derived brain macrophages. Although macrophage-like cells are detected in the rat olfactory epithelium after bulbectomy by using the OX 42 antibody (Suzuki et al., 1995
), the present paper is the first study showing such morphological transformation of microglia-like cells in the peripheral neuroepithelium upon virus infection.
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Acknowledgments |
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References |
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Received 19 February 2002;
accepted 29 April 2002.