Abstract
Neutrophils and macrophages are professional phagocytes that are essential to the immune response and that have developmental and functional similarities. They derive from a common myeloid progenitor and require common transcription factors for their differentiation, such as PU.1 and the CCAAT/enhancer binding proteins C/EBPα and C/EBPε. However, the morphologic maturation of these two lineages is distinct; neutrophil progenitors undergo a progressive series of nuclear changes that culminate in the formation of lobulated nuclei, whereas macrophage progenitors first form circulating monocytes that contain indented nuclei, and then form mature tissue macrophages that contain small, spherical nuclei. To date, little is known regarding the mechanism that regulates nuclear morphogenesis of either lineage. In recent efforts to further understand neutrophil differentiation, we and others have demonstrated that nuclear lobulation in both human and mouse neutrophils is dependent on lamin B receptor (LBR) expression. LBR is an integral membrane protein of the inner nuclear envelope that interacts with B-type lamins and heterochromatin. We have shown that LBR expression is significantly upregulated during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of murine hematopoietic progenitor EML/EPRO cells, which parallels changes in LBR expression exhibited by ATRA-induced human leukemic HL-60 cells. Both models show decreased expression of lamin A/C during neutrophil maturation, whereas lamin B expression remains constant. Importantly, loss of LBR protein expression causes hypolobulation of neutrophil nuclei that is characteristic of the Pelger-Huët anomaly in both mice and humans. These studies support the notion that dynamic changes in the expression of LBR and perhaps lamin A/C play critical roles in neutrophil nuclear maturation. Studies that examine macrophage nuclear maturation, however, have been limited to those performed on HL-60 cells, which showed that LBR expression increased during phorbol ester (TPA) induction. We have now examined the expression of LBR in two additional stem cell factor (SCF)-dependent murine myeloid cell lines that can be induced toward mature neutrophils, pBIM and SCF ER-Hoxb8, as well as in a GM-CSF-dependent ER-Hoxb8 cell line that differentiates into mature macrophages. The pBIM cell line was generated by insertional mutagenesis of mouse marrow cells with the empty pBabe-puro vector. The ER-Hoxb8 cell lines were generated from mouse marrow that was transduced with an estrogen-regulated Hoxb8, which arrests myeloid differentiation. Similar to our findings in EML/EPRO cells, Lbr transcription was upregulated in pBIM cells upon G-CSF-induced neutrophil differentiation, and LBR protein levels significantly increased in SCF ER-Hoxb8 cells upon estrogen withdrawal. By comparison, Lbr transcription dramatically increased during the initial stages of macrophage differentiation of GM-CSF ER-Hoxb8 progenitors, but then decreased in mature macrophages. Initial increases in Lbr transcription during both neutrophil and macrophage differentiation paralleled upregulated expression of both PU.1 and C/EBPε. We also detected increased expression of Lbr in pBIM cells that were generated from a C/EBPε-knockout mouse. This result was surprising given that very recent studies have demonstrated that C/EBPε can directly activate the Lbr promoter. Finally, lamin A/C expression increased during macrophage differentiation of GM-SCF ER-Hoxb8 cells, but was minimally detected in uninduced or differentiated SCF ER-Hoxb8 cells. Together our data indicate that changes in the expression of LBR and lamin A/C play important roles during the maturation of both granulocyte and macrophage lineages, and that PU.1 and/or C/EBPε drive these changes in expression. However, our analyses of C/EBPε-knockout cells indicate that LBR transcription is not absolutely dependent on C/EBPε expression. Current studies to identify the expression and interplay of nuclear envelope proteins during neutrophil and macrophage differentiation that employ these cell line models as well as primary marrow progenitors will further define the mechanisms that regulate morphologic maturation of two important phagocytes.
Lamin A, lamin B, and lamin B receptor analogues in yeast.