Granular cells are required for encapsulation of foreign targets by insect haemocytes

Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. A primary defense response is encapsulation; a process in which haemocytes attach to the foreign organism and kill it. Whether encapsulation requires cooperation between specific subpopulations of haemocytes is unknown. Using purified subpopulations of haemocytes and an in vitro encapsulation assay, we investigated the process of capsule formation in the insect Pseudoplusia includens. Immunocytochemical staining revealed that capsule formation involves a three step process. Encapsulation began when granular cells attached to the foreign target. This was followed by attachment of multiple layers of plasmatocytes. Termination of capsule formation occurred when a subpopulation of granular cells formed a monolayer around the periphery of the capsule. Neither granular cells nor plasmatocytes were capable of forming a capsule independently. However, plasmatocytes encapsulated targets if granular cells were present or if targets were preincubated in medium conditioned by granular cells. The effect of granular cell-conditioned medium could be blocked by the addition of the cell adhesion recognition sequence, RGDS, but not by RGES. These results demonstrate experimentally that granular cells are required for encapsulation of foreign targets by plasmatocytes in vitro, and that the role of granular cells in this process involves an RGD-dependent cell adhesion mechanism.