Transmembrane adaptor proteins are molecules specialized in recruiting cytoplasmic proteins to the proximity of the cell membrane as part of the signal transduction process. A member of this family, SLP65/SLP76, Csk-interacting membrane protein (SCIMP), recruits a complex of SLP65/SLP76 and Grb2 adaptor proteins, known to be involved in the activation of PLC␥1/2, Ras, and other pathways. SCIMP expression is restricted to antigen-presenting cells. In a previous cell line-based study, it was shown that, in B cells, SCIMP contributes to the reverse signaling in the immunological synapse, downstream of MHCII glycoproteins. There it mainly facilitates the activation of ERK MAP kinases. However, its importance for MHCII glycoprotein-dependent ERK signaling in primary B cells has not been analyzed. Moreover, its role in macrophages and dendritic cells has remained largely unknown. Here we present the results of our analysis of SCIMP-deficient mice. In these mice, we did not observe any defects in B cell signaling and B cell-dependent responses. On the other hand, we found that, in dendritic cells and macrophages, SCIMP expression is up-regulated after exposure to GM-CSF or the Dectin-1 agonist zymosan. Moreover, we found that SCIMP is strongly phosphorylated after Dectin-1 stimulation and that it participates in signal transduction downstream of this important pattern recognition receptor. Our analysis of SCIMP-deficient dendritic cells revealed that SCIMP specifically contributes to sustaining long-term MAP kinase signaling and cytokine production downstream of Dectin-1 because of an increased expression and sustained phosphorylation lasting at least 24 h after signal initiation.Dectin-1 is a pattern recognition receptor from the C-type lectin receptor family (1). It is expressed in macrophages, dendritic cells, neutrophils, and a subset of T and B cells (2-4). Through its carbohydrate recognition domain, it specifically recognizes ␤-1,3-glucan (5), which is a typical component of fungal cell walls (6). Dectin-1 is considered a major receptor for ␤-glucans and plays an important role in the defense against various species of pathogenic fungi in mice, including Candida albicans, Aspergillus fumigatus, and Pneumocystis carinii (7-11). The importance of dectin-1 for antifungal defense has also been demonstrated by studies of human patients with disrupted dectin-1 function who display increased mucosal colonization with Candida species and suffer from recurrent mucocutaneous fungal infections (12, 13).Dectin-1 signaling is initiated by phosphorylation of the hemITAM motif in its intracellular tail, leading to the recruitment and activation of the protein tyrosine kinase Syk. This is followed by sequential activation of PLC␥2 and PKC␦. Stimulation of this pathway as well as of additional Syk-independent pathways results in the activation of the transcription factors NF-B, nuclear factor of activated T cells (NFAT), and IRF1/5 and initiation of signaling by the MAP kinases ERK, p38, and JNK, which then contribute to downstr...

The canonical Wnt signaling pathway is mediated by interaction of b-catenin with the TCF/LEF transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of b-catenin that yielded contradictory results. Here, we employed a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) which specifically abrogates b-catenin-TCF/LEF interaction. Disruption of the b-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced G-CSF receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of b-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candidaalbicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of b-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the b-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the b-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2 cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1b by neutrophil granulocytes. In this study, we show that in addition to IL-1b, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2 cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2 cmo mice did not affect IL-1b levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2 cmo mice.

LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes. LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.

The receptor-type protein tyrosine phosphatase CD45 promotes onset and severity of IL-1β-mediated autoinflammatory osteomyelitis

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukaemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which correlates with ETV6-RUNX1 (t(12;21)(p13;q22)) translocation and favourable prognosis in childhood leukaemia. It has a broad expression pattern in haematopoietic and in non-haematopoietic cells. However, its physiological function has been unknown. Here, we show that WBP1L negatively regulates signalling through a critical chemokine receptor CXCR4 in multiple leucocyte subsets and cell lines. We also show that WBP1L interacts with NEDD4-family ubiquitin ligases and regulates CXCR4 ubiquitination and expression. Moreover, analysis of Wbp1l-deficient mice revealed alterations in B cell development and enhanced efficiency of bone marrow cell transplantation. Collectively, our data show that WBP1L is a novel regulator of CXCR4 signalling and haematopoiesis. | 1981BORNA et Al.

Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR) and Fc-receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK), blocks TCR but not BCR signaling. SFK are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK and ZAP-70 deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared to ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels.

IntroductionAutoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo. In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1β and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1.MethodsTo address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers.ResultsOur data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1β production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues.ConclusionsWe demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1β pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.