Figure 4.

Cell adhesion to mindin. (A) α_Mβ₂-mediated cell adhesion to mindin. A total of 2 × 10⁶ α_Mβ₂-expressing or mock-transfected HEK 293 cells were allowed to adhere to 24-well non-tissue culture polystyrene plates precoated with recombinant mindin-FL, mindin-FS or its different mutants. After incubation at 37°C for 30 min, unbound cells were removed by three washes with DPBS and adherent cells were quantified by staining with crystal violet, measuring absorption at 570 nm. Specificity of cell adhesion was verified using the α_Mβ₂-specific antagonist NIF, mock-transfected cells and biotinylated IgG or ovalbumin as the substrate (not shown). Data shown are the means±s.d. of triplicate wells and are representative of three independent experiments. (B) The α_Mβ₂-mediated cell adhesion to mindin is mediated by its α_M I domain. Cell adhesion to mindin-FL by different integrin receptors, including α_Mβ₂, α_Lβ₂, α_L(Iα_M)β₂ and α₂(Iα_M)β₁, where the α_L or α₂ I domain was replaced with that of α_M, were determined as above. Specificity was verified by the addition of the α_Mβ₂-specific antagonist NIF and by using mock-transfected HEK 293 cells. Data shown are the means ±s.d. of triplicate wells and are representative of two independent experiments. (C) Neutrophil adhesion to mindin is enhanced by LPS. Murine neutrophils were added to mindin-coated 24-well plates (0.5 × 10⁶ cells per well) in the presence or absence of 1 μg/ml LPS. After 10 min at 37°C, unbound cells were removed by washing with PBS and bound cells were quantified by crystal violet staining, measuring absorption at 570 nm.