Abstract
Neutrophils and macrophages are critical mediators of innate immunity that share multiple features, including differentiation from common myeloid progenitors that exhibit similar changes in protein expression profiles as they mature into each lineage. Proteins common to both lineages include those critical to phagocyte functions, such as antimicrobial enzymes, cell surface receptors and adhesion proteins. These cells also exhibit similar changes to their nuclear structure, at least during the early stages of differentiation when their nuclei become indented or kidney-shaped. While regulators that control myeloid cell protein expression are well characterized, very little is known about why changes occur to their nuclear shape or how this process is regulated. We previously reported that murine neutrophils with homozygous mutations in the lamin B receptor (Lbr) gene exhibited loss of nuclear lobulation, similar to Pelger-Huët anomaly found in humans with deficient LBR expression. These Lbr-/- neutrophils also exhibited deficient chemotaxis, oxidative bursts and proliferation at the promyelocyte stage, indicating that this inner nuclear envelope (NE) protein influences both nuclear morphology and functional maturation. Based on these results, we have studied possible roles of additional NE components in either neutrophil or macrophage differentiation. We focused on three NE-associated proteins that we found were differentially expressed during the maturation of each lineage, the two isoforms of A-type lamins (lamins A and C) that form intermediate filaments within the nuclear lamina, and a component of the linker of nucleoskeleton and cytoskeleton complex termed Sun2. We show that as mouse neutrophil progenitors mature, they exhibit increased Lbr and Sun2 expression but decreased amounts of lamin A or C, consistent with previous results generated from human myeloblastic HL-60 cells. By comparison, differentiating monocytes exhibit dramatically increased levels of lamin A/C expression but decreased levels of Lbr and Sun2 as they mature into macrophages. We hypothesize that these changes indicate that expression levels of each NE protein must be precisely regulated during normal neutrophil and macrophage differentiation, and therefore aberrant expression of these proteins will impact the maturation of either lineage. Moreover, changes in NE protein expression levels in maturing macrophages may affect functional responses in addition to those that mediate innate immunity, including those that support inflammation. To test this hypothesis, we have manipulated myeloid cell line models of mouse neutrophil or macrophage differentiation to overexpress lamin A, lamin C or Sun2, and have examined how overexpression of each protein affects their morphologic and functional maturation. Our studies demonstrate that overexpression of either lamin A or C causes a severe loss of nuclear lobulation in maturing neutrophils, while they accelerate nuclear maturation in differentiating macrophages. Neutrophils overexpressing each A-type lamin also exhibit deficient chemotaxis and phagocytosis, suggesting that these two responses depend on precise levels of each lamina protein. By comparison, mature macrophages overexpressing either lamin A or C exhibit deficient phagocytosis but increased Mac-1 expression that may lead to enhanced cellular adhesion. We also have preliminary evidence that indicate oxidative bursts produced by either lineage are enhanced by ectopic lamin A or C expression. Our preliminary studies of neutrophils with ectopic Sun2 expression indicate a disruption to GM-CSF-dependent growth at the promyelocyte stage similar to that observed in Lbr-/- promyelocytes, while those that do survive and differentiate exhibit normal nuclear morphology but decreased oxidative bursts. Our continued analyses of these different manipulated models will provide unique knowledge of the influence that NE proteins have on not only myeloid cell nuclear structure but also important functional features not previously known to depend on specific levels of NE protein expression. We predict that these studies will provide a new appreciation for the complexity of NE protein expression patterns in maturing myeloid lineages, and for the important roles that NE proteins perform in orchestrating both nuclear morphologic maturation and key phagocyte functions.
Disclosures
No relevant conflicts of interest to declare.
Spatial distribution of lamin A determines nuclear stiffness and stress-mediated deformation