Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan1
Yoneyama Maternity Hospital, 2-12 Shin-machi, Hachioji, Tokyo 192-0065, Japan2
Author for correspondence: Noboru Uchide. Fax +81 426 76 5738. e-mail uchide{at}ps.toyaku.ac.jp
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Abstract |
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Primary cultured chorion and amnion cells were prepared from human foetal membranes obtained by caesarean section during the month of normal parturition and the cells were cultured in 80% DMEM/F12 (GibcoBRL), 20% heat-inactivated foetal bovine serum (FBS), 120 µg/ml kanamycin sulfate, 120 units/ml penicillin G sodium, 120 µg/ml streptomycin sulfate, 16 µg/ml gentamicin sulfate and 0·3 µg/ml amphotericin B, as described previously (Ohyama et al., 2000 ). IV stock suspension was diluted to the desired concentrations using serum-free medium. Confluent cell monolayers were inoculated with the diluted IV (A/PR/8/34, H1N1) suspension at the desired m.o.i. as described (Fujimoto et al., 2000
). Mock-infected control cells were treated similarly but without virus.
Apoptosis induction was examined by DNA fragmentation assays based on agarose gel electrophoresis as described (Ohyama et al., 1998 ). After mock and IV infection at an m.o.i. of 40, chorion and amnion cells were cultured for various periods. Fig. 1
shows that, in chorion cells, DNA fragmentation into oligonucleosomes was detected at 24 h after IV infection and increased gradually with increased incubation time. DNA laddering occurred in infected chorion cells but not in either mock-infected chorion cells or mock- and IV-infected amnion cells. Since DNA laddering is a key feature of apoptotic cell death (Wyllie et al., 1980
), these results indicated that IV infection induced apoptosis in chorion cells, but not in amnion cells.
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The features of THP-1 cells that caused adherence to a substrate after exposure to IV-C-sup were analysed. THP-1 cells (1·25x105 cells) were incubated for 4 days with 50% IV-C-sup on glass coverslips in 24-well plates in a total volume of 0·5 ml. Alternatively, non-treated THP-1 cells were smeared on to glass slides. Firstly, the morphology of cells stained with Giemsas solution was observed under an Eclipse E600 microscope (Nikon). Non-treated THP-1 cells were round, the nucleocytoplasmic ratio was >1 and the cytoplasm was highly basophilic with a few vacuoles (Fig. 3A). The THP-1 cells exposed to IV-C-sup on coverslips were irregularly shaped, the nucleocytoplasmic ratio was decreased to <1, and the cytoplasm was weakly basophilic with many vacuoles (Fig. 3B
). Next, we examined the phagocytotic function of IV-C-sup-treated THP-1 cells on coverslips. On day 4, the cells were incubated with 1x107 particles/ml of 2 µm fluorescent latex particles (PolySciences, PA, USA) at 37 °C or 0 °C for 4 h as described (Oda & Maeda, 1986
). Fluorescence microscopy revealed many fluorescent latex particles in IV-C-sup-treated THP-1 cells at 37 °C (Fig. 3C
), while particles were virtually absent after the incubation at 0 °C (Fig. 3D
). Furthermore, the number of fluorescent latex particles per cell (200 cells per sample) was counted visually in phase contrast views under the fluorescence microscope. The percentage of cells carrying fluorescent latex particles was 78·0±7·2 (P<0·001) after a 4 h incubation at 37 °C, and 7·8±6·7 after a 4 h incubation at 0 °C. The number of fluorescent latex particles per cell was 5·2±2·1 (P<0·005) after a 4 h incubation at 37 °C, and 0·1±0·1 after a 4 h incubation at 0 °C. The data are shown as means and standard deviations calculated from four independent experiments. Statistical analysis using the t-test (n=4) showed significant differences between incubations at 37 °C and 0 °C. These observations demonstrated that the morphology of IV-C-sup-treated THP-1 cells on the coverslips was characteristic of macrophages (Tsuchiya et al., 1982
), and that the fluorescent latex particles were phagocytosed by the cells in a temperature-dependent manner, suggesting that the THP-1 cells were morphologically and functionally differentiated to macrophages by IV-C-sup. Furthermore, THP-1 cells were cultured with mock-infected chorion cell culture supernatant (mock-C-sup) and IV-C-sup for 4 days on glass coverslips. THP-1 cells that adhered on the coverslips were incubated with fluorescent latex particles at 37 °C for 4 h. In treatments with both mock-C-sup and IV-C-sup, the percentage of cells carrying fluorescent latex particles was between 86·5 and 87·0%, and the mean latex particle number per cell was between 6·1 and 6·3. No significant difference in percentages of THP-1 cells carrying fluorescent latex particles or mean of fluorescent latex particle number per cell was observed between treatments with mock-C-sup and IV-C-sup. Next, THP-1 cells adhered to a coverslip in a microscopic view were counted (magnification x400) after staining with Giemsas solution. The number of THP-1 cells that adhered to a coverslip by treatment with mock-C-sup was 61·6±15·9, while the number with IV-C-sup was 221·8±51·5 (t-test, n=10, P<0·001). A significant difference in the number of THP-1 cells adhered to the coverslips was observed between the treatments. As expected, the total number of fluorescent latex particles phagocytosed with IV-C-sup-treated THP-1 cells was 3·7 times greater than that of mock-C-sup-treated THP-1 cells, indicating that IV-C-sup significantly enhanced phagocytotic reaction by increasing the number of differentiated macrophages.
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In conclusion, the present study has shown that IV-infected cultured human chorion cells undergoing apoptosis released a soluble factor, which was stable at 56 °C for 30 min and which could make monocytes differentiate to macrophages. Furthermore, the expression of SR-A mRNA was induced by factors derived from IV-infected chorion cells undergoing apoptosis. SR-A is implicated in the recognition and clearance of apoptotic cells (Peiser & Gordon, 2001 ), in addition to
v
3 integrin, thrombospondin, phosphatidylserine receptor, CD36 and 61D3 antigen (McCarthy & Evan, 1999
). Therefore, IV-infected apoptotic cells may lead to recognition and clearance of self cells by mature macrophages with SR-A protein. A fibroblast-derived differentiation inducing factor (F-DIF), identical to interleukin (IL)-6, induces the differentiation of monocytic leukaemia cell lines including THP-1 to macrophages, and this activity is synergistically enhanced by respective combination with tumour necrosis factor (TNF)-
, interferon (IFN)-
, granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1 and IL-4 (Noda et al., 1991
; Takeda et al., 1988
). Because IV infection was found to induce the mRNA expression of IL-1
, IL-6, TNF-
, IFN-
and GM-CSF in cultured human chorion cells but not in amnion cells (unpublished data), some of these cytokines might be associated with the differentiation-inducing activity in the culture supernatant of IV-infected chorion cells undergoing apoptosis. Since it is known that TNF-
and IL-6 are heat-stable at 56 °C for 30 min (Ruff & Gifford, 1980
; Fujibayashi & Matsuda, 1991
), these cytokines become the default candidates for the proposed differentiation-inducing factor at present. We are now investigating whether or not cytokines released from IV-infected chorion cells act as putative factors in the differentiation-inducing activity.
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References |
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Received 29 August 2001;
accepted 27 November 2001.
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