| The greater wax moth, Galleria mellonella, is used extensively as a model to study innate immunity. The multicellular process of nodulation acts to isolate large numbers of small microbes (bacteria and yeasts) in the open circulatory system (hemocoel) of the insect and is directed by the main immune blood cell (hemocyte) types, the granular cells and the plasmatocytes. G proteins and their downstream products have been implicated in both early (hemocyte-microbe adhesion) and late (humoral) immune responses, however, their involvement in hemocyte-hemocyte interactions leading to nodule formation is not known. Herein, I treated hemocytes with an AB5 protein, whole cholera toxin (CTX), known to stimulate the Galphas subunit of heterotrimeric G proteins and is responsible for activating adenylate cyclase and elevating intracellular cAMP in mammalian neutrophils. CTX caused a bimodal increase in hemocytes per in vitro microaggregate and nodule frequency in vivo, wherein increasing high cholera toxin concentrations induced these effects, while low concentrations induced levels of hemocytes per in vitro microaggregate and nodule frequency similar to the highest concentration of CTX, but the hemocytes showed reduced substratum-related adhesion. CTX-dependent bacterial (Bacillus subtilis) removal from the insect hemocoel was inversely correlated with nodule frequency. The cholera toxin B-subunit was responsible for the effects of CTX at higher concentrations, while isolated cholera toxin A-subunit had no effect on the hemocytes. In vitro CTX-induced, hemocyte microaggregate formation was inhibited by RGD peptides, suggesting the involvement of integrins.;Human integrin antibodies to alphav, beta3, alpha 5, and beta1 bound integrin-like molecules from unseparated, whole hemocyte lysates and labeled both the granular cells and plasmatocytes adhering to coverslips. The anti-alpha5 integrin labeled the junction between interacting granular cells and plasmatocytes, the frequency of this labeling pattern increasing with CTX treatment. Only anti-alpha 5 and -beta1 prevented Ba. subtilis removal from the insect hemocoel with increasing antibody concentration. |
