Magnesium Dependence of Endotoxin-Induced Degranulation Oflimulusamebocytes

Amebocytes are blood cells that function in the defense system of the horseshoe crab Limulus polyphemus. When they are withdrawn from the animal, they flatten and degranulate within hours. We have found, however, that when a small amount of blood is drawn into a syringe containing a large amount of 3% NaCl, free of divalant cations, and further diluted (to a final concentration of approximately 1:500) in 3% NaCl into tissue-culture-treated plates, amebocytes do not flatten or degranulate, even in the presence of endotoxin. This dilution technique was used to determine direct effects of Ca++ and Mg++ in the flattening and degranulation of amebocytes. Ca++ or Mg++ added at normal hemolymph concentration to 3% NaCl caused rapid flattening. Degranulation did not occur for several days. In the presence of nanogram quantities of endotoxin, cells in Ca++ fortified saline remained granulated, while those in Mg++ fortified saline completely degranulated within several hours. Experiments with EGTA and EDTA confirmed that Ca++ is not essential for degranulation of amebocytes in the presence of serum. Although Ca++ and Mg++ are both influential in the in vitro flattening of Limulus amebocytes, Mg++ is essential for the process of degranulation.