Xu Ming Dai

Langerhans cells arise from monocytes in vivo.

Langerhans cells (LCs) are the only dendritic cells of the epidermis and constitute the first immunological barrier against pathogens and environmental insults. The factors regulating LC homeostasis remain elusive and the direct circulating LC precursor has not yet been identified in vivo. Here we report an absence of LCs in mice deficient in the receptor for colony-stimulating factor 1 (CSF-1) in steady-state conditions. Using bone marrow chimeric mice, we have established that CSF-1 receptor–deficient hematopoietic precursors failed to reconstitute the LC pool in inflamed skin. Furthermore, monocytes with high expression of the monocyte marker Gr-1 (also called Ly-6c/G) were specifically recruited to the inflamed skin, proliferated locally and differentiated into LCs. These results identify Gr-1hi monocytes as the direct precursors for LCs in vivo and establish the importance of the CSF-1 receptor in this process.

Retinoblastoma promotes definitive erythropoiesis by repressing Id2 in fetal liver macrophages

In mammals, the fetal liver is the first site of definitive erythropoiesis—the generation of mature, enucleated red cells. The functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by erythroblasts at various stages of differentiation. Targeted disruption of the retinoblastoma (Rb) tumour suppressor gene in the mouse leads to embryonic death caused by failure of erythroblasts to enucleate. The erythroid defect has been attributed to loss of Rb in cells that support erythropoiesis, but the identity of these cells is unknown. Here we show that Rb-deficient embryos carry profound abnormalities of fetal liver macrophages that prevent physical interactions with erythroblasts. In contrast, wild-type macrophages bind Rb-deficient erythroblasts and lead them to terminal differentiation and enucleation. Loss of Id2, a helix–loop–helix protein that mediates the lethality of Rb-deficient embryos, rescues the defects of Rb-deficient fetal liver macrophages. Rb promotes differentiation of macrophages by opposing the inhibitory functions of Id2 on the transcription factor PU.1, a master regulator of macrophage differentiation. Thus, Rb has a cell autonomous function in fetal liver macrophages, and restrains Id2 in these cells in order to implement definitive erythropoiesis.

The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation

The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r-/-) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II-V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r-/- mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis.

Osteoclast Deficiency Results in Disorganized Matrix, Reduced Mineralization, and Abnormal Osteoblast Behavior in Developing Bone

Studies of the influence of the osteoclast on bone development, in particular on mineralization and the formation of the highly organized lamellar architecture of cortical bone by osteoblasts, have not been reported. We therefore examined the micro‐ and ultrastructure of the developing bones of osteoclast‐deficient CSF‐1R‐nullizygous mice (Csf1r−/− mice).

CSF-1 receptor structure/function in MacCsf1r-/- macrophages: regulation of proliferation, differentiation, and morphology.

CSF‐1 is the major regulator of tissue macrophage development and function. A GM‐CSF‐dependent, CSF‐1 receptor (CSF‐1R)‐deficient F4/80hiMac‐1+Gr1–CD11c+ bone marrow macrophage (BMM) line (MacCsf1r−/−) was developed to study the roles of the eight intracellular CSF‐1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild‐type CSF‐1R rescued the CSF‐1‐induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y → F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF‐1‐inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF‐1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r−/− macrophage line will facilitate genetic and proteomic approaches to CSF‐1R structure/function studies in the major disease‐related CSF‐1R‐expressing cell type.

BCL-6 Negatively Regulates Expression of the NF-κB1 p105/p50 Subunit

BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin’s B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-κB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5′ regulatory region of NF-κB1 in vitro and in vivo, and repress NF-κB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6−/− mice further confirm the biological relevance of BCL-6-dependent regulation of NF-κB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6−/− macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-κB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear κB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-κB1 expression, thereby inhibiting NF-κB-mediated cellular functions.

Colony Stimulating Factor-1 Dependence of Paneth Cell Development in the Mouse Small Intestine

BACKGROUND & AIMS Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development. METHODS CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1. RESULTS Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages. CONCLUSIONS These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohns disease and ulcerative colitis.

Transgenic expression of CSF-1 in CSF-1 receptor-expressing cells leads to macrophage activation, osteoporosis, and early death

CSF‐1 is the primary mononuclear phagocyte and osteoclast growth factor. Autocrine regulation by CSF‐1 has been reported in macrophages during inflammatory responses and in neoplastic cells. To investigate whether inflammatory disease or neoplasia was the dominant consequence of autocrine regulation by CSF‐1 in CSF‐1 receptor (CSF‐1R)‐expressing cells, we created mice that express CSF‐1 under the control of the CSF‐1R promoter/first intron driver [transgene TgN(Csf1r‐Csf1)Ers (TgRC) mice], which have reduced thymic size, a short lifetime, and low body weight and develop osteoporosis. In 4‐week‐old TgRC mice, osteoclast numbers are elevated, and macrophage densities are increased in bone marrow, spleen, liver, and brain. Cultured TgRC macrophages express CSF‐1 and proliferate without exogenous CSF‐1 and in the presence of neutralizing antimouse CSF‐1 antibody. Compared with macrophages from nontransgenic littermates, TgRC macrophages exhibit a stellate morphology, express elevated mRNAs for proinflammatory cytokines, and despite a lower, steady‐state cytokine secretion, secrete elevated levels of inflammatory cytokines in response to LPS, indicating that TgRC macrophages are functionally primed through the CSF‐1R. Thus, autocrine regulation of CSF‐1R‐expressing cells by CSF‐1 leads to a severe phenotype that emphasizes the importance of the known, local production of CSF‐1 in inflammatory disease.

Distinct In Vivo Roles of Colony-Stimulating Factor-1 Isoforms in Renal Inflammation

CSF-1, the major regulator of macrophage (Mφ) development, has three biologically active isoforms: a membrane-spanning, cell surface glycoprotein, a secreted glycoprotein, and a secreted proteoglycan. We hypothesized that there are shared and unique roles of individual CSF-1 isoforms during renal inflammation. To test this, we evaluated transgenic mice only expressing the cell surface or precursors of the secreted CSF-1 isoforms for Mφ accumulation, activation, and Mφ-mediated tubular epithelial cell (TEC) apoptosis during unilateral ureteral obstruction. The only difference between secreted proteoglycan and secreted glycoprotein CSF-1 isoforms is the presence (proteoglycan) or absence (glycoprotein) of an 18-kDa chondroitin sulfate glycosaminoglycan. We report that 1) cell surface CSF-1 isoform is sufficient to restore Mφ accumulation, activation, and TEC apoptosis to wild-type levels and is substantially more effective than the secreted CSF-1 isoforms; 2) the chondroitin sulfate glycosaminoglycan facilitates Mφ accumulation, activation, and TEC apoptosis; 3) increasing the level of secreted proteoglycan CSF-1 in serum amplifies renal inflammation; and 4) cell-cell contact is required for Mφ to up-regulate CSF-1-dependent expression of IFN-γ. Taken together, we have identified central roles for the cell surface CSF-1 and the chondroitin sulfate chain on secreted proteoglycan CSF-1 during renal inflammation.

CSF-1 Receptor-Dependent Colon Development, Homeostasis and Inflammatory Stress Response

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1op/op mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r−/− and Csf1op/op mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r−/− colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r +/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r +/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.