Clorfl86/RHEX Is a Negative Regulator of SCF/KIT Signaling in Human Skin Mast Cells.

Mast cells (MCs) are key effector cells in allergic and inflammatory diseases, and the SCF/KIT axis regulates most aspects of the cells' biology. Using terminally differentiated skin MCs, we recently reported on proteome-wide phosphorylation changes initiated by KIT dimerization. C1orf186/RHEX was revealed as one of the proteins to become heavily phosphorylated. Its function in MCs is undefined and only some information is available for erythroblasts. Using public databases and our own data, we now report that RHEX exhibits highly restricted expression with a clear dominance in MCs. While expression is most pronounced in mature MCs, RHEX is also abundant in immature/transformed MC cell lines (HMC-1, LAD2), suggesting early expression with further increase during differentiation. Using RHEX-selective RNA interference, we reveal that RHEX unexpectedly acts as a negative regulator of SCF-supported skin MC survival. This finding is substantiated by RHEX's interference with KIT signal transduction, whereby ERK1/2 and p38 both were more strongly activated when RHEX was attenuated. Comparing RHEX and capicua (a recently identified repressor) revealed that each protein preferentially suppresses other signaling modules elicited by KIT. Induction of immediate-early genes strictly requires ERK1/2 in SCF-triggered MCs; we now demonstrate that RHEX diminution translates to this downstream event, and thereby enhances NR4A2, JUNB, and EGR1 induction. Collectively, our study reveals RHEX as a repressor of KIT signaling and function in MCs. As an abundant and selective lineage marker, RHEX may have various roles in the lineage, and the provided framework will enable future work on its involvement in other crucial processes.

How "Neuronal" Are Human Skin Mast Cells?

Mast cells are evolutionarily old cells and the principal effectors in allergic responses and inflammation. They are seeded from the yolk sac during embryogenesis or are derived from hematopoietic progenitors and are therefore related to other leukocyte subsets, even though they form a separate clade in the hematopoietic system. Herein, we systematically bundle information from several recent high-throughput endeavors, especially those comparing MCs with other cell types, and combine such information with knowledge on the genes' functions to reveal groups of neuronal markers specifically expressed by MCs. We focus on recent advances made regarding human tissue MCs, but also refer to studies in mice. In broad terms, genes hyper-expressed in MCs, but largely inactive in other myelocytes, can be classified into subcategories such as traffic/lysosomes (MLPH and RAB27B), the dopamine system (MAOB, DRD2, SLC6A3, and SLC18A2), Ca2+-related entities (CALB2), adhesion molecules (L1CAM and NTM) and, as an overall principle, the transcription factors and modulators of transcriptional activity (LMO4, PBX1, MEIS2, and EHMT2). Their function in MCs is generally unknown but may tentatively be deduced by comparison with other systems. MCs share functions with the nervous system, as they express typical neurotransmitters (histamine and serotonin) and a degranulation machinery that shares features with the neuronal apparatus at the synapse. Therefore, selective overlaps are plausible, and they further highlight the uniqueness of MCs within the myeloid system, as well as when compared with basophils. Apart from investigating their functional implications in MCs, a key question is whether their expression in the lineage is due to the specific reactivation of genes normally silenced in leukocytes or whether the genes are not switched off during mastocytic development from early progenitors.

Effect of thrombopoietin receptor agonists on leukocyte and haematopoietic stem and progenitor cells in the peripheral blood of patients with immune thrombocytopenic purpura.

The thrombopoietin receptor agonists (TPO-RAs), romiplostim and eltrombopag, stimulate megakaryopoiesis and thereby increase platelet counts. Both drugs are increasingly used in the treatment of immune thrombocytopenic purpura (ITP). To assess the effect of TPO-RAs on trilineage haematopoiesis, colony-forming cell (CFC) assays were performed on peripheral blood mononuclear cells of 8 healthy donors and 52 ITP patients. Additionally, we revaluated the regular and complete blood counts (CBCs) performed during romiplostim therapy in 45 patients and the CBCs performed in 9 patients during eltrombopag therapy. The clonogenic capacity of PBMCs was significantly increased in patients treated with TPO-RAs compared with healthy donors and untreated patients [BFU-E, 69 ± 47; CFU-GM, 61 ± 48; CFU-GEMM, 16 ± 11; CFU-total, 145 ± 94; P values < 0.05)]. Relative leukocytosis was observed in routine blood counts in 12 of 45 (26%) patients treated with romiplostim. The regular CBCs, performed time dependent within the first 5 days, revealed a maximum increase of leukocytes on days 2 and 3 following romiplostim administration. There were no significant changes in red blood cell parameters. None of the affected patients did recognise any significant symptom, which may be related to leukocytosis. Similarly, we observed a statistically significant increase of leukocyte count in a small cohort of ITP patients (n = 9) in whom CBCs were controlled following treatment initiation (P = 0.044). Our results indicate that TPO-RAs may also mobilize haematopoietic stem and progenitor cells (HSPCs) in peripheral blood and the occurrence of such relative leukocytosis may signalize an early symptom of myelofibrosis due to treatment with TPO-RAs.

A Rare Case of Acute Myeloid Leukemia with a t(2;3) Chromosomal Translocation Characterized by Thrombophilia and Chemoresistance.

We hereby report a case of acute myeloid leukemia with translocation t(2;3) and involvement of the ectopic virus integration site-1 (EVI1) gene. Like most other 3q26-related disorders reported thus far, we describe a phenotype with elevated platelet counts and dysmegakaryopoesis. The clinical course of our patient was complicated by symptomatic thrombophilia and chemoresistance. In addition, our case exhibited FLT3 (Fms-related tyrosine kinase 3) internal tandem duplication. Although anagrelide was successful in controlling elevated platelet counts, allogeneic stem cell transplantation failed to overcome chemoresistance due to simultaneous graft-versus-host-disease and relapse of acute myeloid leukemia. Given the dismal outcome of our case and previously reported cases, we propagate the implementation of targeted therapies to newly diagnosed patients with acute myeloid leukemia t(2;3). Preclinical models indicate drugs that plausibly target the EVI1-related molecular vulnerability as candidates for basket trials. Anagrelide exhibited a hopeful signal of activity in 3q26-related thrombocytosis and should be evaluated for implementation as supportive care.

Treatment of 5 dogs with immune-mediated thrombocytopenia using Romiplostim.

BACKGROUND: Immune thrombocytopenia (ITP) in dogs is analogous to that in humans. Romiplostim, a novel thrombopoietin receptor (TPO-R) agonist, is currently used for the treatment of refractory ITP in humans, but not in dogs. Here, we describe the response to romiplostim in five dogs with refractory ITP. Five dogs with severe and refractory ITP (three primary and two secondary) received romiplostim subcutaneously. Four dogs were administered 3-5 µg/kg and one dog received 10-13 µg/kg body weight once weekly. RESULTS: Romiplostim was well-tolerated and administration was associated with an increase in platelet counts in all five dogs. Four of the five dogs entered remission and relapses were not observed over a follow-up period of 3-10 months. CONCLUSIONS: Romiplostim is effective in the treatment of ITP in dogs at least as well as in humans. This finding may help to develop and use new therapeutics for ITP in dogs and humans.

Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

Proteomic profiling of secreted proteins for the hematopoietic support of interleukin-stimulated human umbilical vein endothelial cells.

Human umbilical cord vein endothelial cells (HUVECs) secrete a number of factors that greatly impact the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). These factors remain largely unknown. Here, we report on the most comprehensive proteomic profiling of the HUVEC secretome and identified 827 different secreted proteins. Two hundred and thirty-one proteins were found in all conditions, whereas 369 proteins were identified only under proinflammatory conditions following IL-1ß, IL-3, and IL-6 stimulation. Thirteen proteins including complement factor b (CFb) were identified only under IL-1ß and IL-3 conditions and may potentially represent HSPC proliferation factors. The combination of bioinformatics and gene ontology annotations indicates the role of the complement system and its activation. Furthermore, CFb was found to be transcriptionally strongly upregulated. Addition of complement component 5b-9 (C5b-9) monoclonal antibody to the stem cell expansion assay was capable of significantly reducing their proliferation. This study suggests a complement-mediated cross-talk between endothelial cells and HSPCs under proinflammatory conditions.

Transcriptional profiling of the hematopoietic support of interleukin-stimulated human umbilical vein endothelial cells (HUVECs).

Endothelial cells can be successfully used to maintain or increase the number of hematopoietic stem cells in vitro. Previously we identified hematopoietic progenitor cell (HPC) expansion or survival benefit induced by IL-1ß-, IL-3-, and IL-6-stimulated human umbilical vein endothelial cell (HUVEC) supernatants. In order to identify molecular mechanisms that support hematopoiesis, we examined the time-dependent expression profiles of IL-1ß-, IL-3-, and IL-6-stimulated HUVECs via microarray. Here, we present 24 common upregulated elements and three common downregulated elements of IL-1ß- and IL-3-stimulated HUVECs, with these factors exhibiting great potential for the observed HPC expansion. Furthermore, metabolic pathway analysis resulted in the identification of nonproteinogenic factors such as prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and determined their HPC expansion potential via delta, methylcellulose, and cobblestone assays. We confirmed PGE(2) and spermine as hematopoietic expansion factors. Furthermore, we identified several factors such as SSAT, extracellular matrix components, microRNA21, and a microvesicle-mediated cross-talk between the endothelium and HPCs that may play a crucial role in determining stem cell fate. Our results suggest that microarray in combination with functional annotations is a convenient method to identify novel factors with great impact on HPC proliferation and differentiation.

Interleukin 32 promotes hematopoietic progenitor expansion and attenuates bone marrow cytotoxicity.

The identification of soluble factors involved in stem cell renewal is a major goal in the assessment of the BM niche. We have previously shown that human endothelial cell (EC) supernatants can induce the proliferation of hematopoietic progenitor cells (HPCs), especially after stimulation with IL-1ß. To identify new potential growth factors, we compared the expression profile of IL-1ß-stimulated ECs over 4, 8 and 16 h with non-stimulated ECs using oligonucleotide microarrays covering more than 46,000 transcripts. Most significant changes were detected after 4 h. Utilization of Gene Ontology annotation for the stimulated EC transcriptome indicated multiple upregulated genes encoding extracellular proteins with a cell-cell signaling function. Using flow cytometry, delta, colony and cobblestone assays, we assessed the proliferative capacities of 11 gene products, i.e. IL-8, IL-32, FGF-18, osteoprotegerin, Gro 1-3, ENA78, GCP-2, CCL2 and CCL20, which are not known to induce HPC expansion. Notably, IL-32 and to a lesser degree osteoprotegerin and Gro 3 significantly induced the proliferation of HPCs. Furthermore, IL-32 attenuated chemotherapy-related BM cytotoxicities by increasing the number of HPCs in mice. Our findings confirm that the combination of microarrays and gene annotation helps to identify new hematopoietic growth factors.

Hematopoietic progenitor cells and interleukin-stimulated endothelium: expansion and differentiation of myeloid precursors.

BACKGROUND: Cytokine-stimulated endothelial cells (EC) propagate hematopoietic progenitor cell (HPC) expansion. However, the effects on the functional capacities of cultured progenitors have not been evaluated. HPC were assessed by flow cytometry, colony and cobblestone assays and long-term cultures (LTC) after culturing in the supernatant of EC stimulated by IL-1beta, IL-3 or IL-6. RESULTS: EC incubation with IL-6 did not improve cell expansion in comparison to non-stimulated EC supernatant, while the HPCs' phenotype and functional capacities were retained. In contrast, IL-1beta and IL-3 stimulation resulted in a 10- and 100-fold increase in cell numbers with more than 90% of these cells being CD33(+). Plating efficiencies and LTC initiating cells were greatest in IL-6 supernatants, whereas the highest numbers of burst-forming units were observed using IL-3. IL-1beta supernatants diminished the number of 5-week cobblestone-areas, whereas the number of 2-week cobblestone areas remained equal to freshly isolated HPC. Fewer 2-week cobblestones and greater amounts of 5-week cobblestones were observed with IL-6 and IL-3. Expanded progenitors from all interleukin conditions were further matured into functional granulocytes. CONCLUSION: IL-1beta and IL-3 stimulated endothelium induces proliferation and differentiation of myeloid precursors, while IL-6 treatment induced a benefit of HPC survival.

High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which platelets are opsonised by autoantibodies and destroyed by macrophages. Therefore, ITP represents a prototype of a B-cell-mediated autoimmune disorder. B-cell activating factor (BAFF) is a member of the tumour necrosis factor family and an important regulator of B-cell development. BAFF levels were determined in serum samples from 53 patients with ITP. Serum BAFF levels in patients with an active ITP were increased when compared with the healthy control group (median 1620 pg/ml vs. 977 pg/ml; P < 0.001). Moreover, immunosuppressive treatment was associated with strongly suppressed BAFF levels (median 629 pg/ml; P < 0.01). In addition, a polymorphic site was detected in the BAFF promoter region (-871) that appeared to occur more frequently in ITP patients than in healthy persons. This promoter variant was associated with very high BAFF levels in ITP patients. Our data suggest that BAFF is an important pathogenetic factor in the development of ITP.