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Fig. 5. Migration of murine neutrophils through laminin-coated filters is dependent on PECAM-1, 6 integrins and NE. 96-well NeuroProbe chemotaxis chambers were used to investigate neutrophil migration through laminin-coated filters. Transmigration filters (3 µm pores) were coated with 2% BSA, laminin-1 (LN-1; 15 µg ml^–1) or laminin-1 coated with a combination of 20% PECAM-1 and 80% ICAM-1 (using a solution with a total protein concentration of 10 µg ml^–1). Bone-marrow-derived mouse neutrophils (2x10⁵) obtained from wild-type or PECAM-1 KO mice were placed on top of the filters with saline or IL-8 (10^–7 M) in the bottom wells. Before addition to filters, cell samples were treated with saline (control), control IgG1b or IgG2a mAbs (both at 10 µg ml^–1; these data were pooled because there was no significant difference between them), a mAb against mouse ß2 integrins (10 µg ml^–1), the mAb GoH3 against 6 integrin (10 µg ml^–1) or the NE-specific inhibitor ONO-5046 (50 µM final concentration), as indicated. After an incubation period of 3 hours at 37°C, the numbers of transmigrated cells in the bottom wells were counted by microscopy after Kimura staining. All samples were run in triplicate and the results represent means±s.e.m. from three to five independent experiments. Statistically significant responses from transmigration through BSA-treated filters are shown by asterisks (**P<0.01). Significant inhibition of transmigration through filters coated with laminin-1/PECAM-1/ICAM-1 in samples treated with GAME 46 or GoH3 compared with control mAb-treated samples are shown by hashes (^#P<0.05) and significant inhibition of response by the NE inhibitor ONO-5046 compared with saline-treated cells is shown by crosses (⁺⁺P<0.01).

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