1 Department of Microbiology, Fukui Medical University School of Medicine, Shimoaisuki 23-3, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan
2 Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan
Correspondence
Yoshinobu Kimura
ykimura{at}fmsrsa.fukui-med.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In the present study, we infected IL-18-gene disrupted (IL-18-/-) mice with influenza A virus intranasally and investigated the host defence mechanism, particularly focusing on the local responses in the respiratory tract. The response of the wild-type C57BL/6 mice to influenza virus infection was used as a control.
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METHODS |
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Mice.
IL-18-/- mice, with the genetic background of C57BL/6 mice, were generated by the gene targeting method (Takeda et al., 1998). The mice were bred and maintained in the Animal Laboratory of Fukui Medical University under specific pathogen-free conditions. The concentration of IL-18 molecules in serum from IL-18-/- mice, measured by ELISA, was below the detectable level (cf. 1000±20 pg ml-1 in normal C57BL/6 mice). This indicated that the IL-18 gene mutation leads to a lack of IL-18 production. Six-week-old IL-18-/- mice and age-matched C57BL/6 mice (Clea Japan) were used for the experiments.
Experimental infections.
Mice were mildly anaesthetized by intraperitoneal administration of pentobarbital sodium (0·025 mg g-1 body mass) and inoculated in the right nostril with influenza A virus in 20 µl sterile PBS. To avoid laboratory contamination, all virus-infected mice were housed in negatively pressurized isolators equipped with a ventilation system through a high-efficiency particulate air filter (AH model; Nihon-Ika). This work was approved by the Committee of Institutional Animal Care and Use in Fukui Medical University.
Preparation of single-cell suspensions from the lung parenchyma.
Mice were anaesthetized and the lung was flushed in situ with 20 ml sterile PBS via cannulation of the heart to remove the intravascular blood pool. Minced lung tissues were incubated at 37 °C for 60 min on a rocker with 200 µg collagenase D ml-1 and 40 µg DNase I ml-1 (both from Roche Molecular Biochemicals). Subsequently, the enzyme-digested lung tissue was passed through a stainless steel mesh. Single-cell suspensions were collected by density-gradient centrifugation with lymphocyte separation solution (Antibody Institute).
Identification of lung parenchyma cells.
Single-cell suspensions from the lung parenchyma were identified by flow cytometry (EPCS XL, Beckman Coulter) using monoclonal antibodies for CD4, CD8, CD16/32 and neutrophil (Caltag).
Assay of cytokine and nitric oxide production in the BAL fluids.
The amounts of cytokines such as IFN-, IL-12 and IL-4 were assayed by using a mouse cytokine detection ELISA kit (BioSource) as described previously (Liu et al., 2003
). The minimum detectable concentrations of IFN-
, IL-12 and IL-4 were 1 pg ml-1, 2 pg ml-1and 5 pg ml-1, respectively. The level of nitric oxide was measured with a Nitric Oxide Assay kit (Calbiochem-Novabiochem), in accordance with the manufacturer's instructions.
Assay of NK cell and cytotoxic T lymphocyte (CTL) activities in the lung parenchyma.
A cytotoxicity assay was performed according to the protocol previously described (Liu et al., 2001). Lung parenchyma cells and NK-sensitive Yac-1 target cells were mixed and incubated at 37 °C in a 5 % CO2 atmosphere for 4 h. Specific lysis of target cells was determined by a lactate dehydrogenase release assay (Decker & Lohmann-Matthes, 1988
) using a Cytotoxic Detection kit (Roche). Data were expressed as the percentage of specific release: 100x[(target with effector - effector spontaneous) - target spontaneous]/[target maximum - target spontaneous]. For the assay of specific CTL activity, target cells were prepared using mouse lymphoma EL-4 cells infected with influenza A virus at an input m.o.i. of 10 p.f.u. per cell.
Antibody assay.
Virus-specific immunoglobulins (Igs) were measured with an ELISA Ig Quantitative kit (Bethyl Laboratories) as described previously (Dong et al., 2003). Bronchoalveolar lavage (BAL) fluids and sera were collected and assayed for a T helper type 2 (Th2)-related antibody, IgG1, and for a Th1-related antibody, IgG2a.
Statistical significance.
The two-tailed MannWhitney U-test and Student's t-test were used to determine whether a significant difference (P<0·05) existed between IL-18-/- and control C57BL/6 mice.
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RESULTS |
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DISCUSSION |
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The results presented in this paper show that, during the early stage of influenza virus infection, just before the appearance of specific immune responses, the reduced NK activity of lung parenchyma cells in IL-18-/- mice was correlated with profound virus growth in the lung. Thus, IL-18 plays an important role in the early antiviral host response by up-regulation of the NK cell-mediated killing activity of virus-infected cells. A similar early antibacterial action of IL-18 has also been found during Streptococcus pneumoniae infection (Lauw et al., 2002).
IFN has long been recognized as an essential part of the innate cytokine response to virus infection (Hennet et al., 1992), showing immunomodulatory activity as well as direct antiviral activity. During the process of IFN-
production, IL-18 acts in synergy with IL-12 (Kohno et al., 1997
). It is interesting to note that, despite the high titre of IL-12 induced in IL-18-/- mice, production of IFN-
was still at a low level (Fig. 5
). This finding implies that IL-12 alone, in the absence of IL-18, is insufficient for activation and development of IFN-
production in vivo and that functions operated by IL-18 molecules cannot fully be compensated by IL-12. The major cell population responsible for IFN-
production by IL-18 stimulation is that of T lymphocytes and NK cells (Hunter et al., 1997
). The lower levels of production of IFN-
in IL-18-/- mice during the early days of infection might be due to a reduction in the number of IFN-
-expressing NK cells (Pien et al., 2000
), although the total number of NK cells in IL-18-/- mice was slightly greater than in wild-type mice (Table 1
).
Pneumonia is characterized by the recruitment of phagocytic cells, mainly granulocytes, to the site of infection (Skerrett, 1994). The influx of granulocytes into the lung alveolar compartment at the early phase of influenza virus infection was markedly increased in IL-18-/- mice (Table 1
). This vigorous recruitment of granulocytes was associated with proinflammatory stimuli (Greenberger et al., 1996
; Tsai et al., 1998
), which might be induced by the heavy loading of virus infection in the lung (Fig. 2
). The enhanced granulocyte influx into the lung is naturally followed by excessive production of nitric oxide, which is generated mostly by granulocytes and is involved in the pathogenesis of influenza virus-induced pneumonia (Akaike et al., 1996
).
No appreciable difference in the local humoral and cellular immune responses upon influenza virus infection could be found between IL-18-/- mice and wild-type mice (Table 2). Even IL-18-/- mice cleared progeny virus from the lung completely without delay when the specific immune responses were developed effectively. These results indicate that IL-18 has no effect on the induction and development of influenza virus-specific immunity but controls the innate NK cell and IFN system at an early stage of infection.
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ACKNOWLEDGEMENTS |
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Received 25 August 2003;
accepted 7 October 2003.