Cloning of rat interleukin-3 receptor β-subunit from cultured microglia and its mRNA expression in vivo

Abstract

The high-affinity receptors for interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of a ligand binding (α-) and a transducing (β-) subunit. Two distinct transducing subunits (clones AIC2A and AIC2B) have been cloned from mouse, whereas in humans, only one (common) β-subunit (β(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 β-subunit in the rat. Most likely, the new rIL-3Rβ cDNA is the rat equivalent of human respective murine (AIC2B) β(c) subunits. Regulation of rIL-3Rβ mRNA expression was investigated in cultured microglia and in vivo. Purified microglia expressed significant amounts of rIL-3Rβ mRNA. Addition of lipopolysaccharide (LPS) resulted in a marked upregulation of rIL-3Rβ mRNA within approximately 4 hr. No downregulation was observed within 1 week's treatment. No rIL-3Rβ mRNA was detectable in normal rat brain. However, 3 hr after a single injection of LPS into the tail vein of a rat, a marked induction of receptor mRNA occurred in a variety of brain regions. Transcriptional rates subsided significantly after 24 hr. rIL-3Rβ mRNA was visualized by in situ hybridizations with CRNA antisense probes in ramified cells formerly characterized as microglial cells. rIL-3Rβ mRNA was also induced in rat brain after occlusion of middle cerebral artery (MCAO). Time course of induction was slower than in lipopolysaccharide (LPS)-treated animals and tasted for more than 24 hr until a significant downregulation became apparent. In centers of infarcted areas, receptor-positive cells likely were blood-borne macrophages and microglia, whereas in areas distant from lesions, cells with morphologies typical of microglia stained positive with digoxigenin (dig)-labeled cRNA probes. It is concluded, that induction of rIL-3Rβ mRNA in brain microglial cells is a very early marker of microglial activation in vivo.