Synergism between IL-33 and MRGPRX2/FcεRI Is Primarily Due to the Complementation of Signaling Modules, and Only Modestly Supplemented by Prolonged Activation of Selected Kinases.

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33's contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules.

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.

CREB Is Activated by the SCF/KIT Axis in a Partially ERK-Dependent Manner and Orchestrates Survival and the Induction of Immediate Early Genes in Human Skin Mast Cells.

cAMP response element binding protein (CREB) functions as a prototypical stimulus-inducible transcription factor (TF) that initiates multiple cellular changes in response to activation. Despite pronounced expression in mast cells (MCs), CREB function is surprisingly ill-defined in the lineage. Skin MCs (skMCs) are critical effector cells in acute allergic and pseudo-allergic settings, and they contribute to various chronic dermatoses such as urticaria, atopic dermatitis, allergic contact dermatitis, psoriasis, prurigo, rosacea and others. Using MCs of skin origin, we demonstrate herein that CREB is rapidly phosphorylated on serine-133 upon SCF-mediated KIT dimerization. Phosphorylation initiated by the SCF/KIT axis required intrinsic KIT kinase activity and partially depended on ERK1/2, but not on other kinases such as p38, JNK, PI3K or PKA. CREB was constitutively nuclear, where phosphorylation occurred. Interestingly, ERK did not translocate to the nucleus upon SCF activation of skMCs, but a fraction was present in the nucleus at baseline, and phosphorylation was prompted in the cytoplasm and nucleus in situ. CREB was required for SCF-facilitated survival, as demonstrated with the CREB-selective inhibitor 666-15. Knock-down of CREB by RNA interference duplicated CREB's anti-apoptotic function. On comparison with other modules (PI3K, p38 and MEK/ERK), CREB was equal or more potent at survival promotion. SCF efficiently induces immediate early genes (IEGs) in skMCs (FOS, JUNB and NR4A2). We now demonstrate that CREB is an essential partaker in this induction. Collectively, the ancient TF CREB is a crucial component of skMCs, where it operates as an effector of the SCF/KIT axis, orchestrating IEG induction and lifespan.

CREB Is Indispensable to KIT Function in Human Skin Mast Cells-A Positive Feedback Loop between CREB and KIT Orchestrates Skin Mast Cell Fate.

Skin mast cells (MCs) are critical effector cells in acute allergic reactions, and they contribute to chronic dermatoses like urticaria and atopic and contact dermatitis. KIT represents the cells' crucial receptor tyrosine kinase, which orchestrates proliferation, survival, and functional programs throughout the lifespan. cAMP response element binding protein (CREB), an evolutionarily well-conserved transcription factor (TF), regulates multiple cellular programs, but its function in MCs is poorly understood. We recently reported that CREB is an effector of the SCF (Stem Cell Factor)/KIT axis. Here, we ask whether CREB may also act upstream of KIT to orchestrate its functioning. Primary human MCs were isolated from skin and cultured in SCF+IL-4 (Interleukin-4). Pharmacological inhibition (666-15) and RNA interference served to manipulate CREB function. We studied KIT expression using flow cytometry and RT-qPCR, KIT-mediated signaling using immunoblotting, and cell survival using scatterplot and caspase-3 activity. The proliferation and cycle phases were quantified following BrdU incorporation. Transient CREB perturbation resulted in reduced KIT expression. Conversely, microphthalmia transcription factor (MITF) was unnecessary for KIT maintenance. KIT attenuation secondary to CREB was associated with heavily impaired KIT functional outputs, like anti-apoptosis and cell cycle progression. Likewise, KIT-elicited phosphorylation of ERK1/2 (Extracellular Signal-Regulated Kinase 1/2), AKT, and STAT5 (Signal Transducer and Activator of Transcription) was substantially diminished upon CREB inhibition. Surprisingly, the longer-term interference of CREB led to complete cell elimination, in a way surpassing KIT inhibition. Collectively, we reveal CREB as non-redundant in MCs, with its absence being incompatible with skin MCs' existence. Since SCF/KIT regulates CREB activity and, vice versa, CREB is required for KIT function, a positive feedforward loop between these elements dictates skin MCs' fate.

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.

ß-arrestin-1 and ß-arrestin-2 Restrain MRGPRX2-Triggered Degranulation and ERK1/2 Activation in Human Skin Mast Cells.

As a novel receptor that efficiently elicits degranulation upon binding to one of its numerous ligands, MRGPRX2 has moved to the center of attention in mast cell (MC) research. Indeed, MRGPRX2 is believed to be a major component of pseudo-allergic reactions to drugs and of neuropeptide-elicited MC activation in skin diseases alike. MRGPRX2 signals via G proteins which organize downstream events ultimately leading to granule discharge. Skin MCs require both PI3K and ERK1/2 cascades for efficient exocytosis. ß-arrestins act as opponents of G proteins and lead to signal termination with or without subsequent internalization. We recently demonstrated that ligand-induced internalization of MRGPRX2 requires the action of ß-arrestin-1, but not of ß-arrestin-2. Here, by using RNA interference, we find that both isoforms counter skin MC degranulation elicited by three MRGPRX2 agonists but not by FcεRI-aggregation. Analyzing whether this occurs through MRGPRX2 stabilization under ß-arrestin attenuation, we find that reduction of ß-arrestin-1 indeed leads to increased MRGPRX2 abundance, while this is not observed for ß-arrestin-2. This led us speculate that ß-arrestin-2 is involved in signal termination without cellular uptake of MRGPRX2. This was indeed found to be the case, whereby interference with ß-arrestin-2 has an even stronger positive effect on ERK1/2 phosphorylation compared to ß-arrestin-1 perturbation. Neither ß-arrestin-1 nor ß-arrestin-2 had an impact on AKT phosphorylation nor affected signaling via the canonical FcεRI-dependent route. We conclude that in skin MCs, ß-arrestin-2 is chiefly involved in signal termination, whereas ß-arrestin-1 exerts its effects by controlling MRGPRX2 abundance.

MRGPRX2-Mediated Degranulation of Human Skin Mast Cells Requires the Operation of Gαi, Gαq, Ca++ Channels, ERK1/2 and PI3K-Interconnection between Early and Late Signaling.

The recent discovery of MRGPRX2 explains mast cell (MC)-dependent symptoms independently of FcεRI-activation. Because of its novelty, signaling cascades triggered by MRGPRX2 are rudimentarily understood, especially in cutaneous MCs, by which MRGPRX2 is chiefly expressed. Here, MCs purified from human skin were used following preculture or ex vivo and stimulated by FcεRI-aggregation or MRGPRX2 agonists (compound 48/80, Substance P) in the presence/absence of inhibitors. Degranulation was assessed by ß-hexosaminidase or histamine release. Phosphorylation events were studied by immunoblotting. As a G protein-coupled receptor, MRGPRX2 signals by activating G proteins; however, their nature has remained controversial. In skin MCs, Gαi and Gαq were required for degranulation, but Gαi was clearly more relevant. Ca++ channels were likewise crucial. Downstream, PI3K was essential for granule discharge initiated by MRGPRX2 or FcεRI. ERK1/2 and JNK were additional participants, especially in the allergic route. Addressing possible points of intersection between early and later events, pERK1/2 and pAKT were found to depend on Gαi, further highlighting its significance. Gαq and Ca++ channels made some contributions to the phosphorylation of ERK. Ca++ differentially affected PI3K activation in FcεRI- vis-à-vis MRGPRX2-signaling, as channel inhibition increased pAKT only when triggered via FcεRI. Collectively, our study significantly extends our understanding of the molecular framework behind granule secretion from skin MCs.

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.

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.

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.

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.

Differentially expressed microRNAs in maternal plasma for the noninvasive prenatal diagnosis of Down syndrome (trisomy 21).

OBJECTIVES: Most developmental processes are under the control of small regulatory RNAs called microRNAs (miRNAs). We hypothesize that different fetal developmental processes might be reflected by extracellular miRNAs in maternal plasma and may be utilized as biomarkers for the noninvasive prenatal diagnosis of chromosomal aneuploidies. In this proof-of-concept study, we report on the identification of extracellular miRNAs in maternal plasma of Down syndrome (DS) pregnancies. METHODS: Using high-throughput quantitative PCR (HT-qPCR), 1043 miRNAs were investigated in maternal plasma via comparison of seven DS pregnancies with age and fetal sex matched controls. RESULTS: Six hundred and ninety-five miRNAs were identified. Thirty-six significantly differentially expressed mature miRNAs were identified as potential biomarkers. Hierarchical cluster analysis of these miRNAs resulted in the clear discrimination of DS from euploid pregnancies. Gene targets of the differentially expressed miRNAs were enriched in signaling pathways such as mucin type-O-glycans, ECM-receptor interactions, TGF-beta, and endocytosis, which have been previously associated with DS. CONCLUSIONS: miRNAs are promising and stable biomarkers for a broad range of diseases and may allow a reliable, cost-efficient diagnostic tool for the noninvasive prenatal diagnosis of DS.

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.

Reduced antioxidant capacities in platelets from patients with autoimmune thrombocytopenia purpura (ITP).

Autoimmune thrombocytopenic purpura (ITP) is characterized by an abnormally low platelet count and bleeding risks. The exact triggering event remains elusive. Oxidative stress may play a role in several autoimmune diseases. A direct link between platelets in ITP and oxidative stress has not yet been addressed. The intracellular platelet antioxidant capacity (AOC) in ITP patients in the active phase (n = 24) and remission (n = 12), and 44 healthy controls were analysed with 2',7'-dichlorodihydrofluorescein diacetate, and in combination with hydrogen peroxide. Enzyme activities (EA) of serum glutathione peroxidase (GPx), glutathione reductase (GRed) and catalase (CAT) were investigated colourimetrically in patients and controls. The AOC of ITP patients in the active phase was drastically reduced, with significantly high mean fluorescence intensity values. Higher GPx activity was observed in both active phase and remission in comparison to healthy controls (p < 0.001), with greater activity observed in active ITP than remission (p = 0.001). However, GRed EA was not elevated indicating that reduced glutathione (GSH) is not comparably recovered as consumed, leading to a decreased bioavailability of GSH and increased oxidative stress. These results suggest that oxidative stress is implicated in active ITP and may play a crucial role in its pathophysiology.

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.