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International Child Health Group, University of Oxford Department of Paediatrics, John Radcliffe Hospital Headington, Oxford, United Kingdom
Doug Hacking, International Child Health Group, University of Oxford, Department of Paediatrics, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK. Dr. Hacking is a Medical Research Council (United Kingdom) Clinical Research Training Fellow. E-mail: doug.hacking{at}imm.ox.ac.uk
Key Words: human type 2 epithelial cells (A549 cells) • interferon-1ß • interferon-
• tumor necrosis factor 
INTRODUCTION
Nitric oxide (NO) has been implicated in the pathogenesis of airway inflammation. Recent studies have shown NO production from human type 2 alveolar epithelial cells (A549) in response to stimulation with unpurified respiratory syncytial virus (RSV), with [1
] and without the addition of exogenous cytokines [2
]. However, RSV-infected A549 cells produce interleukin (IL)-1, IL-1ß, IL-8, and tumor necrosis factor (TNF-) [3
, 4
]. It is not clear from these experiments whether production of NO is because of the presence of contaminating cytokines in the cellular component of the viral preparation or whether the virus itself can induce NO directly.
Human long strain of RSV (A2) was cultured on HEp-2 cells and purified on discontinuous sucrose gradients as described previously [5
]. A549 cells were infected with purified RSV at a multiplicity of infection of 1. UV-inactivated, purified RSV and medium alone were used as controls. After 2 h of incubation at 37°C, the medium was replaced with fresh medium or medium containing human recombinant cytokines [TNF-, 100 ng/ml; IL-1ß, 100 ng/ml; and interferon- (IFN-), 1 ng/ml]. Confirmation of cell infection by RSV was made through staining cultures for viral protein 24 h postinfection using a specific rabbit polyclonal antiserum and through the observation of syncytia formation, which always occurred within 48 h. After 72 h of further incubation at 37°C, the plates were wrapped and stored at -70°C. NO was assayed by measurement of reactive nitrogen intermediates (RNI) in culture supernatants using the Griess reaction after reduction of nitrate to nitrite as described previously [6
]. IFN-, TNF-, and IL-1ß were quantitated in cell-culture supernatants by double-antibody (sandwich) enzyme-linked immunosorbent assay kits (R&D Systems, Abingdon, UK), according to the manufacturer’s protocol. The sensitivity of detection of the IFN-, TNF-, and IL-1ß assays were 15.6 pg/ml, 15.6 pg/ml, and 3.9 pg/ml, respectively. All data are summarized as means with standard errors. Differences in mean RNI values were determined using Student’s t-test. A value was considered significant if below 0.05 for two-sided P values.
RNI concentration was assayed in the supernatant of cells cultured in media alone (control), cells infected with purified RSV alone, cells treated with cytokines, and from cells infected with purified RSV, which were also treated with exogenous cytokines. As shown in Figure 1 , A549 cells infected with purified RSV alone produced little RNI; however, this was significantly more RNI than uninfected controls (111.3 nM/µg, SE 25.2; 65.3 nM/µg, SE 4.9, respectively; P=0.01). When purified RSV-infected cells were incubated with cytokines, they produced significantly more RNI than uninfected cells incubated with cytokines alone (1371.5 nM/µg, SE 107.3; 647.2 nM/µg, SE 43.8, respectively; P=<0.001). The addition of inactivated RSV to A549 cells cultured in the presence of cytokines or in media alone did not increase RNI production further (unpublished results).
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, IL-1ß, and IFN- were assayed in the supernatant of cells cultured in media alone, cells infected with purified RSV, and from cells treated with inactivated RSV. TNF-, IL-1ß, and IFN- were not detected in the supernatant in cells cultured in media alone or from cells stimulated with inactivated RSV. Although neither IL-1ß nor IFN- was detected from purified, RSV-treated A549 cell supernatants, TNF- was found (as shown in Fig. 2
) at 48 h (mean 88.6 pg/ml, SE 51.6) and 72 h (mean 165.9 pg/ml, SE 37.7).
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and IL-1ß during virus purification, because no contaminating IL-8, IFN-, TNF-, or IL-1ß was detected in the sucrose-purified viral preparation. In contrast, unpurified RSV preparations grown on HEp-2 cells showed evidence of TNF- (15.6 pg/ml) and IL-1ß (6 pg/ml). We note that it has been shown previously that as little as 10 pg/ml TNF- or IL-1ß could induce IL-8 and intercellular adhesion molecule-1 expression [3
, 4
]. These findings are consistent with differences in the production kinetics of cytokines such as IL-8 from A549 cells that have been noted between unpurified and purified RSV [7
, 8
]. However, when exogenous cytokines were added to A549 cells, we observed that NO production was increased substantially by infection with purified virus. Previously, synergistic induction of NO production has been demonstrated for combinations of cytokines [9
] and for unpurified RSV with cytokines [1
]. These data suggest that the ability of RSV to induce NO production by respiratory epithelial cells may depend more on a cytokine cascade than on a direct stimulatory effect of the virus.
Received December 13, 2001; revised February 2, 2002; accepted February 4, 2002.
REFERENCES
mediates enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus Am. J. Respir. Cell Mol. Biol. 13,602-609[Abstract]
in respiratory syncytial virus-infected pulmonary epithelial cells in vitro Immunology 95,501-506[Medline]
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, P = 0.01; 
, P = <0.001.