These studies document a diverse and extensive difference in the patterns of gene expression between monocytes and macrophages. Greater that 10% of the probe sets employed were differentially expressed between monocytes and macrophages. We employed an unbiased approach, since there are well-recognized differences between resident or inflammatory monocytes and macrophages, depending on the tissue and the environment [2
]. The changes observed were not random, and the differences identified were related to characteristic functions of macrophages. Of particular note, this study documented extensive up regulation of genes related to lipid, fatty acid and steroid metabolism, and the reduction of genes contributing to transcriptional regulation.
M-CSF promotes the differentiation of macrophages from circulating monocytes [5
], but factors in serum, in addition to M-CSF, are required for optimal differentiation [24
]. M-CSF was highly expressed at 16 hours, and was detected in the culture supernatants at 24 and 48 hours, consistent with earlier observations [25
]. Therefore, early in the process of differentiation M-CSF is expressed, and it is capable of enhancing differentiation. These observations identify a positive feedback loop where by the process of differentiation promotes the local induction of M-CSF and its receptor CSF1R. For technical reasons we were not able to knock down genes prior to 48 hours. Mice deficient in c-fms (CSF1R) or M-CSF (op/op) demonstrate deficient development of monocytes [6
]. Nonetheless, our data clearly document the importance of M-CSF and CSF1R in some, but not all, aspects of macrophage differentiation. It is also possible the differentiation was promoted through the firbonectin receptor [27
], since the fibronectin gene was highly induced in macrophages, and fibronectin is secreted by macrophages [27
Additionally, αv, which may serve as a vitronectin or fibronectin receptor, was strongly induced at 16 and 168 hours, while its binding partner integrin β3 was enhanced at 16 hours. Additionally, integrins α5 and β1, which also bind fibronectin, were highly expressed in monocytes, and both increased significantly, but less than 2 fold, in macrophages. Therefore, following the initial adherence of monocytes, the process of differentiation may have been promoted by the expression of both M-CSF [4
] and fibronectin [27
A recent study also examined monocyte to macrophage differentiation, however, they employed the addition of exogenous M-CSF and coated the culture dishes with FCS, rather than using an initial adherence [28
]. Nonetheless, the extent of the changes, and many of the patterns observed in the two studies were similar. For example both studies noted an increase of genes related to lipid and fatty acid metabolism in macrophages and the reduction of genes induced during classical activation of macrophages activation, such as CXCL8, TNFα, IL-1β, and CD69. Additionally, in both studies, macrophage scavenger receptor 1 (MSR1) and the mannose receptor (MRC1) were increased in unactivated macrophage, as well as in alternatively activated M2 macrophages [28
]. There were differences noted between the two methods of differentiation. In both studies the transcriptional expression of chemokine receptors CCR2, CCR7 and CX3CR1 was increased, although changes in CCR5 were different between the two studies. Further there were differences in genes related to regulation of prostaglandins. Overall, these observations demonstrate marked and consistent differences in the transcriptional profile between circulating monocytes and in vitro differentiated macrophages, employing both methods of differentiation.
A unique characteristic noted early in the process of differentiation was the expression of chemokine genes, including CCL2, CCL3, CCL4, CCL7, CCL24, and CXCL5 which are capable of attracting leukocytes, particularly monocytes [29
]. CCR1, but not other chemokine receptors, was also transiently up regulated. These changes returned to baseline by 168 hours, and suggest that as monocytes begin to differentiate, they signal for the recruitment of additional leukocytes. Since both CD14+CD16+ and CD14+CD16− cells were included, we can not determine whether inflammatory or resident monocytes [2
] may respond differently. Additionally, at this early time point, MHC class II molecules, DR, DQ and DP, were down regulated. The reduction of MHC class II molecules at this time suggests that early during differentiation the ability to present antigen is diminished.
The categories of genes regulated in patterns B and C demonstrated unique and overlapping categories that provide insight into the functional diversity that occurs during the process of differentiation. The genes up regulated as identified by patterns B1 and C1 relate to known functions of macrophages including metabolic pathways for lipid, fatty acid and steroid, carbohydrate, and amino acid metabolism, while those down regulated include those related to the mRNA transcription and apoptosis. Additionally, pattern C1 was also represented by genes identified in the molecular function ontogeny, including oxyreductase, reductase, hydrolase, dehydrogenase, transferase, glycosidase, lyase, protease, and acyltransferase. Many of the genes in the hydrolase molecular function, together with others identified by Medline search, were enriched in genes critical for osteoclast differentiation and function, including tartrate resistant acid phosphatase 5, cathepsins D, K, L and L2, and a large number of ATPase, H+ transporting lysosomal proteins. However, the cells were not osteoclasts, since they did not express calcitonin receptor.
Of the biologic processes identified by patterns B1 and C1, the lipid, steroid and fatty acid metabolism pathway was among the most prominent. Macrophage differentiation in the absence of an additional signal, such as the addition of oxidized LDL, resulted in an extensive modulation of genes that are important in the development of atherosclerosis. Proteins important in lipoprotein homeostasis such as APOE, APOC1 and 2 were highly induced (8 to 20 fold) in macrophages, consistent with another study that employed the serial analysis of gene expression method, which utilized short sequence tag oligonucleotides, and M-CSF [30
]. Receptors important for the binding of modified lipoproteins such as MSR1, SCARB2, and CD68 were up regulated during macrophage differentiation, while others such as CD36 (SCARB3), LDLR and lectin-like oxidized receptor (LOX-1), LDL related protein 1(LRP1, APOER) were already highly expressed in monocytes and were not significantly increased in macrophages.
Additionally, genes important in the metabolism and transport of long chain fatty acids including FABP 3, 4, and 5, cholesterol 27-hydroxylase, which regulates the export of cholesterol [31
], lipase A (LIPA) which promotes chylomicron formation and atherosclerosis [32
], and HMGCR, the target of statins [33
] were all up regulated in macrophages. LPL, which may contribute to foam cell formation and hydrolyzes triglycerides in chylomicrons, VLDL and HDL and lysosomal phospholipase A2, which is an independent risk factor of coronary heart disease [34
] were also increased during macrophage differentiation. Further, both ACAT1 and ACAT2, which esterify cholesterol, promoting lipid droplet formation [35
], were increased during macrophage differentiation. However, some genes important in the formation and function of foam cells such as the ABC transport proteins ABCA1 and ABCG1, important for the export of cholesterol and oxidized phospholipids [36
], were highly expressed in monocytes and changed less than 2 fold in macrophages. However, ABCA1 was up regulated in classically, compared to alternatively activated, macrophages [28
]. Both ABCA1 and ABCG1 may also be induced by oxysterols mediated through the LXRα [37
]. Overall, these observations demonstrate that macrophage differentiation, even in the absence of exogenous M-CSF or oxidized LDLs, directs macrophages toward foam cell development.
Transcriptional repression in eukaryotes has been less well studied than transcriptional activation, partially due to the notion that it is more economical to turn on genes than to keep them off [19
]. Our data demonstrate a marked reduction of the expression of transcription factors during macrophage differentiation. Of the classical transcription factors and the nuclear receptors that regulate transcription, almost 80% of the genes identified were down regulated in macrophages. Among the genes down regulated were three members of nuclear receptor subfamily 4 (NR4A1–3), AML1 and AML3 (RUNX1 and 3), C/EBPδ, ETS2, IRF1, FOSB, FOS, JUNB, JUND, MAFF, HIF1A, FOX03A and FOX01A. While many of these genes had been considered important for macrophage development, our observations suggest that these genes may contribute to the maintenance of monocytes, and that down-regulation allowed the monocytes to differentiate. Supporting this concept, both AML-1 and AML-3, which may function by recruiting co-repressors and histone deacetylases, are involved in the transcriptional repression and gene silencing, such as silencing CD4 during T cell development [38
]. Supporting a role of AML-1 in maintaining the monocyte phenotype, the forced reduction of AML-1 by antisense oligonucleotides, or over expression of dominant negative form of AML-1 induced the expression of non-specific esterase, a marker of differentiated macrophages [40
It is of note that many nuclear receptors were down regulated during the differentiation, especially the NR4A1–3 (Nurr77, Nurr1, and NOR-1). They have been identified as immediate-early genes induced in vascular smooth muscle and endothelial cells by growth factors such as platelet derived growth factor and vascular endothelial cell growth factors, and they are found in atherosclerotic lesions [41
]. Therefore, NR4A receptors may contribute to maintaining the monocyte phenotype or for differentiation in the context of a pathologic environment. Cooperation between p57kip2
and NR4A2 contributes to the differentiation of dopaminergic neural cells from stem cells [42
]. LXRα (NR1H3) is the only nuclear factor, which was up regulated during monocyte to macrophage differentiation. This observation is consistent with previous finding that LXRα was expressed during GM-CSF-induced monocyte to macrophage differentiation [43
]. LXRα is important for macrophage survival, suppression of macrophage activation and the export of cholesterol by regulating the expression of ABCA1 and ABCG1 [44
]. Given the extent of the repression of the transcription factors, it was surprising that Kruppel-like factors (KLF 2, 4, 5, 6, 10 and 11) were all reduced, since KLFs may serve as transcriptional repressors of certain genes [46
]. It is possible that they serve as transcriptional repressors in monocytes. The observation that ERG1 and 3 are repressed during macrophage differentiation appears at odds with earlier studies that suggested ERG1 was essential for macrophage differentiation [49
]. However, a recent study demonstrated normal macrophage differentiation in Erg1, 2 or 3 deficient mice [51
]. Therefore, the differential regulation of nuclear receptor genes and the repression of KLF and ERG family members may be important for macrophage differentiation and function.
While the vast majority of transcription factors that changed were down regulated, a number were up regulated including MAF, CYP5 and C/EBPα. Our data demonstrates the potential importance of C/EBPα, since the forced reduction of C/EBPα suppressed the induction of TREM2 and LPL, which were induced after 16 hours. Even though C/EBPα may be important in some aspects of macrophage differentiation, reduction of C/EBPα failed to reduce the expression of NR1H3 (LXRα) or to modify the morphology of the differentiating macrophages. Supporting the importance of C/EBPα, fetal livers deficient in C/EBPα demonstrated an impaired ability to generate macrophages [52
]. Additionally, committed T cell progenitors were reprogrammed to macrophages by the ectopic expression of C/EBPα [53
]. Together, these observations suggest that although repression of the transcriptional program may contribute to monocyte to macrophage differentiation, C/EBPα may be important for this process.