FES KINASE SIGNALING PROMOTES MAST CELL RECRUITMENT TO TUMOURS

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Systemic recruitment of osteoblastic cells in fracture healing

We hypothesise that following a bone fracture there is systemic recruitment of bone forming cells to a fracture site. A rabbit ulnar osteotomy model was adapted to trace the movement of osteogenic cells. Bone marrow mesenchymal stem cells from 41 NZW rabbits were isolated, culture-expanded and fluorescently labelled. The labelled cells were either re-implanted into the fracture gap (Group A); into a vein (Group B); or into a remote tibial bone marrow cavity 48 h after the osteotomy (Group C) or 4 weeks before the osteotomy was established (Group D), and a control group (Group E) had no labelled cells given. To quantify passive leakage of cells to an injury site, inert beads were also co-delivered in Group B. Samples of the fracture callus tissue and various organs were harvested at discrete sacrifice time-points to trace and quantify the labelled cells. At 3 weeks following osteotomy, the number of labelled cells identified in the callus of Group C, was significantly greater than following IV delivery, Group B, and there was no difference in the number of labelled cells in the callus tissues, between Groups C and A, indicating the labelled bone marrow cells were capable of migrating to the fracture sites from the remote bone marrow cavity. Significantly fewer inert beads than labelled cells were identified in Group B callus, suggesting some of the bone-forming cells were actively recruited and selectively chosen to the fracture site, rather than passively leaked into the circulation and to bone injury site. This investigation supports the hypothesis that some osteoblasts involved in fracture healing were systemically mobilised and recruited to the fracture from remote bone marrow sites. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Mesenchymal stem cells in immunoregulation

Mesenchymal stem cells (MSCs) reside within the bone marrow cavity and serve as a reservoir for the continuous renovation of various mesenchymal tissues. Recent efforts suggest that MSCs modulate the immune reactions in vitro and escape the immune surveillance in vivo. We provide herein a discussion of the issues including the current research progress on the in vitro interactions of MSCs with multiple subsets of immune cells (dendritic cells, T cells, B cells and natural killer cells), in vivo transplantation outcomes, the possible underlying mechanisms, future research directions as well as potential clinical implications.

Multi-potent mesenchymal stromal cells in blood.

Peripheral blood-derived multi-potent mesenchymal stromal cells circulate in low number. They share, though not all, but most of the surface markers with bone marrow-derived multi-potent mesenchymal stromal cells, possess diverse and complicated gene expression characteristics, and are capable of differentiating along and even beyond mesenchymal lineages. Although their origin and physio-pathological function are still unclear, their presence in the adult peripheral blood might relate to some interesting but controversial subjects in the filed of adult stem cell biology, such as systemic migration of bone marrow-derived multi-potent mesenchymal stromal cells and the existence of common hematopoietic-mesenchymal precursors. In this review, current studies/knowledge about peripheral blood-derived multi-potent mesenchymal stromal cells is summarized and the above-mentioned topics are discussed.

High concentrations of dexamethasone suppresses the proliferation but not the differentiation of further maturation of human osteoblast precursor in vitro: relevance to glucocorticoid-induced osteoporosis

Objective. The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). Methods. Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 µm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. Results. At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the 2 and 4, but not ß1, integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. Conclusions. A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like prolferation, differentiation and death in vitro

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula in mol% P2O5 (50)-CaO (50-X)-Na2O (X), where X was either 2, 4, 6, 8 or 10 were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion, proliferation and increased cell death in both cell types studied. There was no significant difference in %cell death between the PBGs which was significantly greater than the controls. However, compared to other PBGs, a greater number of cells was found on the 48 mol% CaO which may have been due to either increased adherence, proliferation or both. This composition was capable of supporting osteogenic proliferation and early differentiation and supports the notion that chemical modification of the glass could to lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

THE IMPACT OF VIRUS INFECTION IN DEREGULATION OF CYTOKINE PRODUCTION UPON SECONDARY BACTERIAL INFECTION

Dendritic cells (DCs) secrete cytokines such as interleukin-23 (IL-23) when stimulated with certain Toll-like receptor (TLR) agonists and infected with pathogens such as P. aeruginosa. IL- 23 is a proinflammatory cytokine that plays a critical role in the proliferation and differentiation of the IL-17 producing Th17- CD4 T helper cells. The lack of efficient cytokine production from antigen-presenting cells, such as DCs, can impact CD4 differentiation and thus impair the immune responses against pathogens. Clearance of some bacterial infections, such as Klebsiella pneumonia and Listeria monocytogenes has been shown to be dependent on the induction of IL-23 and therefore, deregulation of these cytokines as a direct result of virus infection may impede immune responses to secondary infections. Here, an inhibition of TLR ligand or P. aeruginosa-induced IL- 23 expression in Lymphocytic Choriomeningitis Virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs) has been demonstrated, indicating that an important function of these cells is disrupted during virus/bacterial coinfection. While production of TNF-α was unaffected in LPS stimulated cells, TNF-α was significantly inhibited in bacterium infected cells by LCMV. Type I IFN in LPS or LCMV infected cell was not detected and therefore, ruling out the possibility of cytokine suppression by Type I IFN. The production of IL-10 was high in BMDCs infected with LCMV and stimulated with LPS or bacteria. Analysis of multiple cytokines produced in this coinfection model demonstrated that LCMV infection impacts specific cytokine production upon LPS or bacterium infection, which may be important for bacterial clearance. This data is important for future immunotherapy use in viral/bacterial coinfection scenarios.

Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state.

Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin(+) cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin(+) DCs in the skin DLNs. Our results show that in contrast to the current view, langerin(+)CD8(-) DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin(+) DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin(+) DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin(+) DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.

The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.

Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)-mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin(+), skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin(+) dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin(+), skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin(+) DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin(+) DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.

Cutting edge: Langerin+ dendritic cells in the mesenteric lymph node set the stage for skin and gut immune system cross-talk.

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer\'s patch-null mice but abolished in the Peyer\'s patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.

The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels

Idiopathic erythrocytosis (IE) is characterized by erythrocytosis in the absence of megakaryocytic or granulocytic hyperplasia, and is associated with variable serum erythropoietin (Epo) levels. Most patients with IE lack the JAK2 V617F mutation that occurs in the majority of polycythemia vera patients. Four novel JAK2 mutant alleles have recently been described in patients with V617F-negative myeloproliferative disorders presenting with erythrocytosis. The aims of this study were to assess the prevalence of JAK2 exon 12 mutations in IE patients, and to determine the associated clinicopathological features.

The classification and diagnosis of erythrocytosis

An absolute erythrocytosis is present when the red cell mass is raised and the haematocrit is elevated above prescribed limits. Causes of an absolute erythrocytosis can be primary where there is an intrinsic problem in the bone marrow and secondary where there an event outside the bone marrow driving erythropoiesis. This can further be divided into congenital and acquired causes. There remain an unexplained group idiopathic erythrocytosis. Investigation commencing with thorough history taking and examination and then investigation depending on initial features is required. Clear simple criteria for polycythaemia vera are now defined. Those who do not fulfil these criteria require further investigation depending on the clinical scenario and initial results. The erythropoietin level provides some guidance as to the direction in which to proceed and the order and extent of investigation necessary in an individual patient. It should thus be possible to make an accurate diagnosis in the majority of patients.

Langerhans cell (LC) proliferation mediates neonatal development, homeostasis, and inflammation-associated expansion of the epidermal LC network.

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.