Macrophage differentiation: A heterogeneous theory

Functional heterogeneity of macrophage populations has been characterized however, questions remain regarding the mechanisms underlying the development of this heterogeneity. A procedure was developed to molecularly phenotype colonies of bone marrow-derived macrophage(s) (BMDM). Gene expression from individual clones was monitored using the reverse transcription polymerase chain reaction (RT-PCR) to permit simultaneous amplification of multiple specific gene transcripts. Internal "nested" primers were utilized in addition to the two traditional external primers thus, increasing the reliability of amplification. Results revealed hierarchal expression of macrophage-associated genes. Predominant colony phenotypes observed were unique both for the period of differentiation and choice of hematopoietic stimulus. Furthermore, colonies were subcloned into different growth factors and phenotyped on sequential days demonstrating that individual clones were not committed to specific phenotypes but retained the capacity to express different functional phenotypes.Tumor necrosis factor-$\alpha$ (TNF$\alpha$) transcripts were present in all colonies suggesting a role for this molecule during macrophage differentiation. Antisense oligomers to the initiation region of TNF$\alpha$ translation were utilized to inhibit TNF$\alpha$ expression and thus, determine its role in BMDM differentiation. GM-CSF-derived cells isolated on day 3 were exclusively vulnerable to inhibition of TNF$\alpha$ expression, displaying a 30% increase in proliferation over control values. Recombinant TNF$\alpha$ was able to rescue antisense-treated cells preventing increased proliferation. Furthermore, exogenous murine TNF$\alpha$ (mTNF$\alpha$) inhibited proliferation and differentiation of CSF-1-derived BMDM 50% while both human (hTNF$\alpha$) and mTNF$\alpha$ induced differentiation of GM-CSF-derived BMDM by 42%.Two distinct receptors for TNF (TNFR; designated p60 and p80) are distinguished by their species specificity for TNF$\alpha$ binding. TNFR p60 binds both hTNF$\alpha$ and mTNF$\alpha$ whereas p80 exclusively binds mTNF$\alpha$. Northern blot analysis demonstrated similar expression of p60 and p80 in CSF1- and GM-CSF-derived BMDM. Results suggest differential signalling in GM-CSF- and CSF-1-derived macrophages with p80 functioning in TNF$\alpha$-mediated growth inhibition of CSF-1-derived progenitors and p60 functioning in TNF$\alpha$-induced differentiation of GM-CSF-derived BMDM.U of I OnlyETDs are only available to UIUC Users without author permissio