FIGURE 2.

Adhesion of primary human keratinocytes and HaCaT keratinocytes to collagen XXIII depends on integrin α₂β₁ but is independent of RGD-binding integrins or glycosaminoglycans. A, ion- and folding-dependent attachment of primary human keratinocytes to collagen XXIII. Serial dilutions of native or heat-denatured collagen XXIII ectodomain (0.3–20 μg/ml) were immobilized on microtiter plates. Human keratinocytes were allowed to attach to the substratum for 45 min in the absence (untreated) or presence of either Mg²⁺/Mn²⁺ or EDTA, followed by staining with crystal violet. Relative adhesion is presented as ΔE (measured extinction minus nonspecific cell attachment to BSA). B, ion-dependent attachment of HaCaT cells to collagen XXIII. Serial dilutions of collagen XXIII ectodomain (0.3–20 μg/ml) were immobilized, and HaCaT keratinocytes were allowed to attach to the substratum for 30 min. Detection and calculation of the relative adhesion ΔE as described above. C, modulation of HaCaT adhesion to collagen XXIII by function blocking antibodies directed to the indicated integrin subunits. HaCaT keratinocytes were preincubated with monoclonal antibodies AIIB2, P1E6, or P1B5 at the indicated concentrations and subsequently allowed to attach to microtiter plates coated with collagen XXIII ectodomain (5 μg/ml). ab, antibody. D, the contribution of RGD-binding integrins and the influence of glycosaminoglycans to HaCaT attachment on collagen XXIII was analyzed by preincubating the cells and microtiter plates with the indicated concentrations of RGD peptide or heparin. HaCaT keratinocytes and microtiter plates coated with collagen XXIII ectodomain (5 μg/ml) were preincubated with the indicated concentrations of heparin. For the experiments shown in C and D, microtiter plates were coated with collagen XXIII ectodomain (5 μg/ml). Attachments were analyzed in the presence of 2 mm Mg²⁺ and 1 mm Mn²⁺, and the values are presented as percentages of inhibition in comparison with noninhibited controls. Nonspecific cell attachment to BSA was subtracted from all of the values. All of the data represent the mean values ± S.D. (n = 3).