RGD-containing molecules induce macropinocytosis in ascidian hyaline amoebocytes

Abstract

Phagocytes of the compound ascidian Botryllus schlosseri are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the leading edge. This gives rise to the formation of initially irregular vesicles which then move to the inside of the cells and acquire a more regular morphology. Both phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD): this suggests that, as in mammals, integrin activation is involved in the induction of both cell spreading and endocytosis. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion, as indicated by nitroblue tetrazolium reduction, and ATP, as indicated by increased cytochrome oxidase activity. On the whole, our results indicate the conservation of common mechanisms of MP induction throughout the Chordate phylum.

Introduction

Macropinocytosis (MP)¹ is a receptor- and clathrin-independent type of fluid-phase endocytosis, characterised by the formation of large (up to 5 μm in diameter) endocytotic vesicles which originate at sites of membrane ruffling. It permits the uptake of small particles, and bacteria as well as the entry of adenovirus (Meier and Greber, 2004, Meier et al., 2002); it is also involved in the ingestion of necrotic but not apoptotic cells (Krysko et al., 2003).

MP has been reported in many cell types and its diffuse occurrence indicates that, in addition to nutrient uptake, it contributes to other cellular processes such as growth and motility (Dharmawardhane et al., 2000). In mammals, constitutive MP occurs in specialised cells involved in immunosurveillance, such as macrophages, neutrophils and dendritic cells (Swanson and Watts, 1995, Nichols and Lippincott-Schwartz, 2001); in other cell types it can be induced by growth factors, such as epidermal growth factor, platelet-derived growth factor, macrophage colony-stimulating factor, interleukin-4, interferon-γ, or phorbol esters (Amyere et al., 2000, Amyere et al., 2002, Araki et al., 1996, Bruewer et al., 2005, Haigler et al., 1979, Hewlett et al., 1994, Racoosin and Swanson, 1992, Sallusto et al., 1995, Steinmann and Swanson, 1995).

Ascidian hyaline amoebocytes (HA) are motile, circulating phagocytes able to recognise and ingest foreign cells or particles (Cima et al., 1996, Cima et al., 2001, Goodbody, 1974, Hirose et al., 2003, Ohtake et al., 1994, Sawada et al., 1993, Smith, 1970, Sugino et al., 1993, Wright, 1981, Zhang et al., 1992). They differ from granular amoebocytes which are much less abundant, have a cytoplasm filled with numerous granules, are not phagocytic and belong to another differentiation line (Ballarin and Cima, 2005). As a consequence of engulfment, they modify their spreading morphology to become globular and less active macrophage-like cells (Ballarin et al., 1993, Ballarin et al., 1994, Ballarin et al., 2002, Cima et al., 1996). Marked cytoskeletal re-organisation, mainly in its actin constituent, and a respiratory burst are associated with ascidian phagocytosis (Ballarin et al., 1994, Cima and Ballarin, 2000). In the compound ascidian Botryllus schlosseri, in vitro ingestion of foreign particles is greatly enhanced by Arg-Gly-Asp (RGD)-containing proteins or peptides in the culture medium (Ballarin et al., 2002). The tripeptide represents a recognition motif for integrins, suggesting the involvement of these adhesion molecules in the interaction of phagocytes with foreign particles. Given the reported role of integrins in mediating MP (Aderem and Underhill, 1999, Zhou et al., 2001), we wondered whether the presence of RGD-containing peptides can stimulate MP in Botryllus phagocytes. Results indicate a remarkable increase of phagocyte spreading and endocytosis, in the form of MP, as well as activation of the respiratory burst.