CLONING OF RAT INTERLEUKIN-3 RECEPTOR BETA-SUBUNIT FROM CULTURED MICROGLIA AND ITS MESSENGER-RNA EXPRESSION IN-VIVO

The high-affinity receptors for interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of a ligand binding (alpha-) and a transducing (beta-) s ubunit. Two distinct transducing subunits (clones AIC2A and AlC2B) hav e been cloned from mouse, whereas in humans, only one (common) beta-su bunit (beta(c)) has been found. A PCR-based cloning strategy was used to obtain a full-length cDNA sequence from rat microglia including 5'- untranslated regions. Sequence analysis revealed a number of features indicative of the presence of only one beta-subunit in the rat. Most l ikely, the new rIL-3R beta cDNA is the rat equivalent of human respect ive murine (AIC2B) beta(c) subunits. Regulation of rIL-3R beta mRNA ex pression was investigated in cultured microglia and in vivo. Purified microglia expressed significant amounts of rIL-3R beta mRNA. Addition of lipopolysaccharide (LPS) resulted in a marked upregulation of rIL-3 R beta mRNA within approximately 4 hr. No downregulation was observed within 1 week's treatment. No rIL-3R beta mRNA was detectable in norma l rat brain. However, 3 hr after a single injection of LPS into the ta il vein of a rat, a marked induction of receptor mRNA occurred in a va riety of brain regions. Transcriptional rates subsided significantly a fter 24 hr, rIL-3R beta mRNA was visualized by in situ hybridizations with cRNA antisense probes in ramified cells formerly characterized as microglial cells, rIL-3R beta mRNA was also induced in rat brain afte r occlusion of middle cerebral artery (MCAO). Time course of induction was slower than in lipopolysaccharide (LPS)-treated animals and laste d for more than 24 hr until a significant downregulation became appare nt. In centers of infarcted areas, receptor-positive cells likely were bloodborne macrophages and microglia, whereas in areas distant from l esions, cells with morphologies typical of microglia stained positive with digoxigenin (dig)-labeled cRNA probes, It is concluded, that indu ction of rIL-3R beta mRNA in brain microglial cells is a very early ma rker of microglial activation in vivo. [Key words: microglia, inferleu kin-3, receptor, fever induction, middle cerebral artery (IMCAO) RT-po lymerase chain reaction, RACE]