Yasuhiro Waki

Flow cytometric detection of cell-associated cytokines in alveolar macrophages

To elucidate the cytokine-producing capacity of alveolar macrophages (AMs), we have introduced a method of flow cytometry combined with saponin treatment to detect the cell-associated cytokines. We studied bronchoalveolar lavage fluid cells from six patients with sarcoidosis (SAR) and six control (CTL) subjects. Cells were either left uncultured, or cultured with and without lipopolysaccharide (LPS), then treated with paraformaldehyde and saponin and analysed for cell-associated interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) by flow cytometry. Cell-associated IL-1 beta and TNF-alpha were also analysed by immunoassays. The flow cytometric cytokine values were correlated with the immunoreactive cell-associated cytokines (IL-1 beta: r = 0.45, p < 0.05; TNF-alpha: r = 0.82, p < 0.001). The histograms of cell-associated IL-1 beta yielded a single peak both in the patients and controls, whereas the histograms of cell-associated TNF-alpha exhibited two peaks in SAR patients, but just a single peak in the CTL subjects. The mean value of the cell-associated TNF-alpha in LPS+ AMs was higher in the SAR patients than in the CTL subjects (p < 0.001). In conclusion, the flow cytometric method can be applied to the semiquantitative detection of cell-associated cytokines in alveolar macrophages at the single cell level.

Augmentation of endotoxin-induced pulmonary responses by mononuclear cell phagocytosis in the reticuloendothelial system.

OBJECTIVE To test the hypothesis that the effects of intravenous injection of latex particles would demonstrate the contribution of phagocytosis by mononuclear phagocytes to the development of Escherichia coli-induced acute lung injury in neutropenic guinea pigs. DESIGN Prospective, controlled, experimental study. Intravenously injected the latex particles into 41 guinea pigs to investigate the contribution of the phagocytosis in acute lung injury. SUBJECTS Forty-one guinea pigs. INTERVENTIONS Forty-one guinea pigs were divided into five experimental groups: a saline group (n=9); an endotoxin group (n=10) receiving 2 mg/kg of intravenous E. coli endotoxin; a latex group (n=7) receiving 2 x 10(9)/kg of intravenous polystyrene latex (mean diameter 3.19 micrometers); an endotoxin + latex group (n=8); and an E. coli group (n=7) receiving 2 x 10(9) live E. coli/kg. MEASUREMENTS AND MAIN RESULTS The lung wet/dry ratio was increased in the live E. coli-treated guinea pigs (6.71 +/- 0.16 [SEM], p < .01) as compared with the saline control (5.40 +/- 0.16, whereas the ratio was not increased in the endotoxin (5.52 +/- 0.14) or latex (5.58 +/- 0.20) groups. However, the lung wet/dry ratio was greater in the endotoxin + latex group (6.11 +/- 0.16, p < .05) than in the saline control. The 125I albumin lung tissue/plasma ratio was greater in the E. coli (2.00 +/- 0.29, p < .01) and endotoxin + latex (0.84 +/- 0.12, p < .05) groups than in the saline group (0.18 +/- 0.07), whereas no increases were observed in the endotoxin group (0.22 +/- 0.10) and the latex (0.34 +/- 0.13) group. More than 40% of the injected radiolabeled latex was observed to have accumulated in the reticuloendothelial system (liver and spleen), in both the saline control (40.1 +/- 2.3%, n=4) and endotoxin (57.3 +/- 6.8%, n=5) groups, with 2.6 +/- 1.5% and 3.1 +/- 1.7% in the lungs for the saline control and the endotoxin groups, respectively. The percent deposition of radiolabeled latex in the liver was greater in the endotoxin group (51.7 +/- 3.8%, p < .05) than in the saline group (37.6 +/- 5.9%). CONCLUSIONS These findings suggest that, in neutropenic guinea pigs: a) the combination of endotoxin and latex particles induces acute lung injury; and b) the phagocytic properties of mononuclear phagocytes in the reticuloendothelial system augment endotoxin-induced pulmonary responses and may play a role in the development of live E. coli-induced acute lung injury.