DIFFERENTIAL EXPRESSION OF NOVEL GENES BY BONE-MARROW-DERIVED MACROPHAGE POPULATIONS

In the present study, we have constructed a subtraction cDNA library t o identify novel genes induced by IFN-gamma in GM-CSF-derived bone mar row macrophages (ma). Mo were treated with 50 U/ml IFN-gamma for 40, 7 0 and 140 min to induce expression of early genes regulated by IFN-gam ma, and the ms were pooled. Poly(A)(+)RNA was prepared from both unact ivated and IFN-gamma-stimulated mo, and cDNA libraries were constructe d in lambda ZAP. Genes expressed in common by both ms populations were removed by subtraction using biotin-avidin precipitation of hybrid co mplexes. Further selection was performed by differential screening usi ng cDNA prepared from mRNA of unactivated ms as a probe, followed by c olony hybridization to remove sister clones. Of 17 clones from which s equence information was obtained, two appeared to be identical with th e murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an addit ional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match pu blished sequences were used to probe Northern blots prepared from both unstimulated and IFN-gamma-activated GM-CSF- and CSF-1-derived mo. Fi ve clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced tran script levels following IFN-gamma treatment of GM-CSF-derived ms, but demonstrated high constitutive transcript levels in CSF-1-derived mo. In addition, C10 transcripts were constitutively expressed by GM-CSF-d erived ms, but not by CSF-1-derived mo, even after activation by IFN-g amma. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-l-derived mo resides in the differential expression of early genes specifically induced by IFN-gamma.