Microscopical, Biochemical, and Immunological Studies of the Immune Defense System of the Horseshoe Crab, Limulus polyphemus

¹ University Clinic for Infectious Diseases, Endotoxin Laboratory, Rigshospitalet, Copenhagen, Denmark
² Department of Anatomy and Cytology, University of Odense, Campusvej 55, 5230 Odense M, Denmark

The immune defense system of Limulus polyphemus was explored. Two distinct cells, granulocytes and plasmatocytes, were revealed in the hemolymph by light microscopy of live cells, by light microscopy serial sectioning, and by transmission electron microscopy combined with immuno-gold labelling. The granulocytes (amebocytes) comprised about 99% of the hemocytes. They had a heterochromatic nucleus, distended but poorly developed RER, few free ribosomes, few mitochondria, but many large secretory granules. While the majority of these were uniformly stained, structured granules were also present. Monoclonal antibodies revealed that coagulogen is present in both types of large secretory granules. It is suggested that a structured granule is an immature stage leading to a uniformly stained granule. The plasmatocytes had an euchromatic nucleus, extended RER consisting of tubular or flattened cisternae, many free ribosomes, many mitochondria, but few, if any, large secretory granules. Coagulogen was not detected in plasmatocytes. Following immunizations of five rabbits for more than one year with cell-free hemolymph, 14 polypeptides were immunogenic in rabbits as visualized by crossed immunoelectrophoresis. However, O'Farrell gel electrophoresis combined with silver staining identified more than 70 major polypeptides in the cell-free hemolymph. At least 60 of these had pI-values between 7.5 and 5.5, and Mr-values from larger than 200 x 10³ to smaller than 21 x 10³.