Identification of an opioid peptide secreted by rat embryonic mixed brain cells as a promoter of macrophage migration

Conditioned media from embryonic mixed cells from the rat brain were used i n a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS ) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17-CM). Based upon c heckerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow-derived macrophages. The implication of a peptide compound in this activity was suggested by its res istance to heat as well as acid treatments, and by its sensitivity to amino peptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI-174 re duced the migration of BM in response to E17-CM by 60%. This migratory acti vity was no longer effective when pertussis toxin-treated BM were used. Whe n the chemotactic effects of selective opioid agonists were compared to tha t of E17-CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indi cated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met-enkephalin-like reactivit y was found by RIA in the E17-CM. Altogether, these observations suggest th at a delta-like opioid peptide released from embryonic mixed brain cells co uld be responsible for the infiltration of the developing CNS by macrophage s precursors.