Cytokines are polypeptide mediators released by a variety of activated immune and nonimmune cells. These key immunoregulatory molecules have critical effects on cells of the immune system. Consideration of the parallels between innate defenses in vertebrates and host defenses of invertebrates led us to test the hypothesis that invertebrate cytokine-like molecules regulate responses to infection or wounding. Interleukin (IL)-1 and IL-6 are two cytokines that are critical immune regulators that participate in every aspect of the vertebrate immune system. Our previous studies have demonstrated the presence of IL-1 and IL-6-like molecules in deuterostome invertebrates. We studied the hemolymph of Manduca sexta and whole body extracts of Calliphora sp. to determine whether protostome invertebrates possess cytokine-like molecules as well.Manduca hemolymph or Calliphora whole body extracts were prepared and then fractionated by gel sieve chromatography. Fractions were assayed for IL-1 and IL-6 activity. Activity was detected in column samples with a M r of 20-30,000 Da. This peak was fractionated by isoelectricfocusing and resulted in the isolation of active fractions with pI's of 5.0, 5.5, and 7.2. These values are similar to vertebrate and echinoderm IL-1 (M r of 20,000 and pI's of 5.0 and 7.2) and IL-6 (M r of 25,000 and a pI of 5.5) molecules. Western blot analysis using polyclonal rabbit antisera to either human IL-1 or IL-6 detected a band with a M r of 19,500 (IL-1) and a band with a M r of 29,500 (IL-6) in the Manduca hemolymph. The Manduca IL-1-like molecule was inhibited by antisera to human IL-1. These results demonstrate that insects possess cytokine-like molecules. This research is supported by the NIH (AI 39685-01).