Department of Microbiology, Fukui Medical University School of Medicine, Shimoaizuki 23-3, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, 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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Recently, an oligodeoxynucleotide (ODN) containing unmethylated cytidine-guanosine (CpG) motifs has been demonstrated to be an excellent immune stimulator to induce a strong Th1 immune response (Chu et al., 1997; Davis, 2000
). It is compelling to investigate whether the CpG ODN is capable of improving immunosenescence in the elderly through energizing the Th1 response. In the present study, we adopted a newly developed senescence-accelerated mouse (SAM) system (Takeda et al., 1997a
). The senescence-prone strain of SAM-P1 mice shows various signs of rapid ageing, such as a shortened lifespan of about half that of ordinary control mice, wrinkled skin and age-dependent geriatric disorders (Takeda et al., 1997b
). The storage and function of CD4+ T-cells of SAM-P1 mice are altered, displaying the Th2-biased status of immune responses and the consequently increased susceptibility to influenza virus infection (Dong et al., 2000
). Prolonged infection with influenza virus in immunocompromised, aged mice has also been observed (Bender et al., 1991
). Herein, we show that treatment of SAM-P1 mice with CpG ODN repairs age-associated immune dysfunction and thereby the mice are saved from lethal influenza virus infection.
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METHODS |
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CpG ODN.
Nuclease-resistant phosphorothioate-modified CpG ODNs, such as ODN 1826, ODN IL-12p40, control non-CpG ODN (Hokkaido System Science), ODN g10 and ODN CG-30 (Nisshinbo), were dissolved in endotoxin/pyrogen-free PBS. Sequence data of these ODNs are listed in Table 1 (CpG motifs are underlined).
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Antibody assay.
Virus-specific immunoglobulins (Igs) were measured with an ELISA Ig Quantitative kit (Bethyl Laboratories). Briefly, microtitre plates were coated with 20 µg potassium tartrate-purified influenza virus at 4 °C overnight. After blocking with 1 % BSA for 30 min, serum samples were added into the well and incubated for 1 h. Bound antibodies were reacted with goat horseradish peroxidase-labelled anti-mouse IgG, IgG1, IgG2a or IgA. Plates were read at 490 nm after the addition of O-phenylenediamine. Antibody titres were calculated through a standard curve made from the reference serum using the calculation software SPECTRA MAX 250 (Molecular Devices).
Assay of CTL and natural killer (NK) cell activities.
Spleen cells were obtained from mice that had been primed with influenza virus 3 weeks before. Lymphocytes were collected through density-gradient centrifugation with lymphocyte-separation solution (Antibody Institute) and re-stimulated in vitro by co-cultivation for 5 days with syngeneic spleen cells that had been infected with influenza virus 1 h before. Target cells were prepared using mouse L929 cells infected with influenza virus at an input m.o.i. of 10 p.f.u. per cell. Effector lymphocytes and target cells were mixed and incubated for 6·5 h at 37 °C. Specific lysis of target cells was determined by lactate dehydrogenase-release assay (Decker & Lohmann-Matthes, 1988) using a Cytotoxicity Detection kit (Roche). Data were expressed as the percentage of specific release: 100x[(target with effector -effector spontaneous) - target spontaneous]/(target maximum -target spontaneous). NK cell activity was measured by specific lysis of Yac-1 cells.
Flow cytometry analysis.
B-cells were doubly stained using the FITC-labelled monoclonal antibody (mAb) for CD19 (Beckman Coulter) and the phycoerythrin (PE)-labelled mAb for CD40. T-cells were stained using the PE-labelled antibodies for CD3, CD4 and CD8 (Caltag) and the FITC-labelled mAb for CD154 (Serotec). The fluorescence intensity of cell samples was assayed on a fluorescence-activated cell sorter (EPICS XL, Beckman Coulter), acquiring 10 000 events per sample. Data were analysed using the computer program SYSTEM 2, version 1.0.
Statistical analysis.
Data represent the mean±SD. Fisher's exact test, the Student t-test or the MannWhitney U-test were used to determine whether a significant difference (P<0·05) existed between the groups tested. Statistical analyses were performed using STATVIEW J4.11 (SAS Institute).
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RESULTS |
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In response to influenza virus infection, spleen cells of SAM-P1 mice displayed the Th2-type dominant cytokine production profile when compared with that of normal mice (Dong et al., 2000). An additional treatment of SAM-P1 spleen cells with ODN 1826 increased the ratio of the titre of IFN-
to that of IL-4, showing a shift to the Th1-type response (Table 2
).
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DISCUSSION |
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Unmethylated ODNs with certain flanking bases of CpG motifs are virtually absent in the vertebrate DNA. This fact suggests that the immunostimulatory actions of CpG ODN is an evolutionary phenomenon whereby the CpG ODN acts as a danger signal that the innate defence system of the vertebrate can recognize and respond to (Krieg et al., 1996). Immunostimulatory CpG ODN exerts a number of stimulatory effects on NK cells (Yamamoto et al., 1992
) and antigen-presenting cells (APCs) such as macrophages, dendritic cells and B-cells (Sparwasser et al., 1998
; Hartmann & Krieg, 2000
). The cellular and molecular mechanisms of the CpG ODN action are evidenced as follows: (1) upregulation of proinflammatory cytokines like IL-6, IL-12 and IFN-
; (2) upregulation of cell surface molecules of MHC, CD40, CD80 (B7-1), CD86 (B7-2) and intracellular adhesion molecule (ICAM)-1; and (3) activation of classic signal transduction pathways such as the stress kinase pathway and the NF-
B pathway (Hacker et al., 1998
). CpG ODN has little capacity for inducing direct effects on either CD4+ or CD8+ T-cells and functions through a Th cell-independent pathway (Cho et al., 2000
). However, a current report has demonstrated that phosphorothioate CpG ODN stimulates thymocytes directly (Mannon et al., 2000
). The expression of MHC class II molecule and adhesion molecule ICAM-1 on dendritic cells and B-cells is impaired in SAM-P1 mice, resulting in the age-related dysfunction of APCs (Haruna et al., 1995
). Furthermore, SAM-P1 mice show a unique deficiency in the cell number of CD4+ T-cells (Dong et al., 2000
) and in the expression of the CD154 (CD40 ligand) molecule (Fig. 3
). Because the CD40CD40 ligand interaction is crucial for the IL-12-dependent priming of Th1 cells (Cella et al., 1996
), it is conceivable that the age-associated impairment of CD40CD40 ligand expression is a causative factor of immunosenescence. CpG ODN treatment of SAM-P1 mice induced a quick response of the increased expression of the CD40 ligand on CD4+ T-cells (Fig. 3
), thus establishing protective Th1 immunity.
This finding suggests another novel mechanism involved in T-cell activation by CpG ODN. Although there appear to be multiple defects in immune function of the elderly and the mechanisms are complicated, restoration of the defective Th1 immune response is the most important strategy for protecting the elderly from infection. CpG ODN has been found to be a potent stimulator of a Th1 response that is effective against many types of pathogens. Thus, CpG ODN treatment, especially of elderly individuals, becomes a promising remedy for infectious diseases and age-related immune disorders.
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ACKNOWLEDGEMENTS |
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Received 6 December 2002;
accepted 3 February 2003.