In their encounters with foreign intruders, the cells of the insect innate immune system, like those of the mammalian immune system, exhibit both humoral and cell-mediated responses. Some intruders can be dispatched by the humoral immune system alone, but many must be phagocytosed by individual hemocytes or encapsulated by interacting hemocytes. Surface proteins of hemocytes control the abrupt transition of hemocytes from resting, nonadherent cells to activated, adherent cells during these cell-mediated responses. Two of these surface proteins, an integrin and a tetraspanin, interact during this adhesive transition. As demonstrated with a hemocyte adhesion assay and a surface plasmon resonance assay, the large extracellular loop of tetraspanin D76 binds to a hemocyte-specific integrin of Manduca sexta. The interaction between the large extracellular loop domain and hemocyte-specific integrin is interrupted not only by a monoclonal antibody (MS13) that binds to a domain of ␤-integrin known to be a ligand-binding site for cell adhesion but also by double-stranded ␤-integrin RNA. Transfected S2 cells expressing tetraspanin mediate adhesion of hemocytes. A monoclonal antibody to tetraspanin D76 perturbs the cell-mediated immune response of encapsulation. These studies involving antibody blocking, RNA interference, and binding assays imply a trans interaction of integrin and tetraspanin on hemocyte surfaces.

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Melanization is a complex innate immune response in insects, involving multiple proteases in the prophenoloxidase (proPO) activation cascade. Hemolymph protease‐1 (HP1), a clip‐domain serine protease expressed in hemocytes, is found constitutively in the Manduca sexta hemolymph, yet its function is still unknown. RNAi techniques have been unsuccessful as a means to study the protease in vivo. As a result, we created a model by using the Drosophila S2 cell system to study the protease. The HP1 cDNA was cloned and incorporated into the S2 cellular genome. The cells were selected, and a stable cell line was established. Western blot analysis indicated that the expression of proHP1 in the cell culture was highly efficient. When the stable cells were treated with dsRNA against HP1, the expression of HP1 was blocked. To test whether HP1 might influence the activation of hemolymph proPO, the stable HP1 S2 cells treated with or without dsRNA were cultured as a monolayer. After induction of HP1 gene expression of the stable cells, M. sexta plasma was added to the culture. We found that the culture media of the cells expressing the HP1 protein turned dark‐colored, with an increase of PO activity, whereas the culture media of the cells in which the HP1 gene was silenced by RNAi remained unchanged. This result suggests that HP1 may function in the melanization/PO activation cascade. (supported by NIH grant no. GM41247)