Sensitivity of Alveolar Macrophages to Substrate Mechanical and Adhesive Properties Sophie Fe ´re ´ ol, Redouane Fodil, Be ´ atrice Labat, Ste ´phane Galiacy, Vale ´rie M. Laurent, Bruno Louis, Daniel Isabey,* and Emmanuelle Planus Inserm UMR 651, Fonctions Cellulaires etMole´culaires de l’Appareil Respiratoire et des Vaisseaux, Equipe Biome´canique Cellulaire et Respiratoire et Universite´Paris XII,Faculte´de Me´decine,Institut Supe´rieur des Biosciences de Paris, Cre´teil, France In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on sub- strates of increasing Young’s modulus: (i) a monolayer of alveolar epithelial cells representing the supple (0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high interme- diate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD- coated beads and corrected for actual bead immersion). Macrophage shape dra- matically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligi- ble effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized nu- merical model describing how low cell height enhances the substrate-stiffness-de- pendence of the apparent (measured) AM stiffness. Altogether, these results sug- gest that macrophages are able to probe their physical environment but the mecha- nosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity. Cell Motil. Cytoskeleton 2006. ' 2006 Wiley-Liss, Inc. Key words: alveolar epithelial cells; cell/substrate stiffness; F-actin; magnetic twisting cytometry; mechanotransduction; prestress INTRODUCTION Pulmonary alveoli are lined by epithelium, com- posed of several cell types, such as type II alveolar epi- thelial cells, on which Alveolar Macrophages (AMs) spread, adhere and migrate. AMs are the principal resi- dent free cells of the distal airspaces and are responsible for maintaining a bacteria and particle free environment *Correspondence to: Daniel Isabey, Inserm UMR 651, Faculte ´ de Me ´decine - 8, rue du Ge ´ne ´ral Sarrail, 94010 Cre ´teil Cedex, France. E-mail: daniel.isabey@creteil.inserm.fr Received 15 November 2005; Accepted 28 February 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cm.20130 ' 2006 Wiley-Liss, Inc. Cell Motility and the Cytoskeleton (2006)