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Fig. 8. TNF-
-induced 3D scattering is dependent on 2ß1 integrin. (A-C) TNF- increases the adhesion of 2A4 cells to collagen-coated dishes. 2A4 cells were harvested from confluent cultures following a 48-hour preincubation in the absence (A) or presence (B) of 10 ng ml-1 TNF- in defined medium and were seeded into collagen- or gelatine-coated bacteriological dishes. After a 60 minute incubation at 37°C, nonadhering cells were washed away and adhered cells were fixed and photographed using an inverted photomicroscope. Nonspecific adhesion to dishes that had not been coated with collagen was negligible. Scale bars, 100 µm. (C) Quantification of cell adhesion. Five randomly selected fields were photographed in each dish and the number of attached cells per field was counted on positive prints. Results represent the mean number of cells per field ± s.e.m. from three independent experiments. Mean values were compared using Student's unpaired t test. *P<0.001 versus control values; **not significantly different from control values. (D,E) TNF- increases cell surface expression of the 2 integrin subunit. Cells were trypsinized and stained with monoclonal antibody against the mouse 2 subunit followed by secondary FITC-conjugated antibody, and were analysed by flow cytometry. (D) The plots show 2A4 cells incubated with monoclonal antibody against the 2 integrin subunit (grey profile) or secondary antibody alone (open profile) after 48 hours without (top) or with (bottom) treatment with 10 ng ml-1 TNF-. The flow-cytometric plots shown are representative of five independent experiments. (E) Cells were treated with TNF- and incubated with monoclonal antibody against 2-integrin subunit as described in D. Mean fluorescence intensities ± s.e.m. from five independent experiments were normalized to secondary antibody fluorescence. Mean values were compared using Student's unpaired t test. P<0.005 for values of TNF- versus control. (F-H) Blocking antibodies to ß1 integrins abrogate TNF--induced 3D scattering in a concentration-dependent manner. Cells were grown in collagen gels in defined medium for 3 days and were subsequently incubated for an additional 6 days with 10 ng ml-1 TNF- in the absence (F) or presence (G) of function-blocking antibody against ß1 integrin (10 µg ml-1). (H) Dose-response analysis of the effect of increasing concentrations of anti-ß1-integrin antibody. An isotype-matched irrelevant (anti-TNP) antibody was used as a control (open column). Quantification of 3D scattering was carried out after 6 days of treatment. Data represent the mean number of single cells per photographic field ± s.e.m. from at least three independent experiments. *P<0.025 compared with cultures incubated with TNF- alone; **P<0.0005 compared with cultures incubated with TNF- alone. (I,L) The 2ß1-integrin antagonist rhodocetin abolishes TNF--induced 3D scattering. (I) Cells were grown in collagen gels in defined medium for 3 days and subsequently incubated for an additional 6 days with 10 ng ml-1 TNF- in the presence of 200 nM rhodocetin. (L) Dose-response analysis of the effect of increasing concentrations of rhodocetin. *P<0.0125 for 50 nM rhodocetin compared with cultures incubated with TNF- alone. (F,G,I) Scale bars, 100 µm.
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