On adaptor proteins shaping Ca2+ signals in activated B lymphocytes

Neumann K. On adaptor proteins shaping Ca2+ signals in activated B lymphocytes. Bielefeld (Germany): Bielefeld University; 2008.Signale des B-Zell-Antigenrezeptors (BZR) regeln sowohl die Aktivierung pathogenspezifischer B-Lymphozyten als auch die Eliminierung selbstreaktiver B-Lymphozyten. Das Ergebnis der BZR-Stimulation kann von Apoptose bis zu Proliferation der B-Lymphozyten reichen. Unpassende Signale können deshalb zu einer Unausgewogenheit zwischen der humoralen Immunität und Selbsttoleranz führen. Folglich hängt die B-Lymphozyten-Aktivierung von einem komplizierten Wechselspiel zwischen positiven und negativen Signalelementen ab. Der Anstieg der zytosolischen Kalziumionen (Ca2+)-Konzentration ist ein zentraler Schritt in der B-Lymphozyten-Aktivierung. Die zeitliche Form des Ca2+ Signals trägt zur differentiellen Aktivierung von Transkriptionsfaktoren bei, welche dann das Schicksal der Zelle beeinflussen. Das Adaptorprotein Grb2 hemmt BZR-vermittelte Ca2+-Mobilisierung. Während meiner Arbeit identifizierte Björn Stork in unserem Labor Dok-3 als ein weiteres Adaptorprotein, das Ca2+ Mobilisierung in B-Zellen hemmt. In dieser Arbeit habe ich die molekularen Mechanismen der hemmenden Funktion der zwei Adaptorproteine untersucht. Dafür wurden DT40 B-Zellen verwendet, deren Gene für Grb2 oder Dok-3 inaktiviert worden sind. Damit konnte gezeigt werden, dass beide Proteine die Aktivierung des Ca2+-mobilisierenden Enzyms PLC-[gamma]2 durch Btk hemmen. Dies zeigte sich durch eine erhöhte BZR-vermittelte Phosphorylierung von PLC-[gamma]2 an einer spezifischen Btk-Phosphorylierungsstelle in der Abwesenheit von Dok-3 oder Grb2. Dieser Befund wurde durch erhöhte IP3-Produktion und einen stärkeren Grad der Entleerung der intrazellulären Ca2+-Speicher nach BZR-Stimulation unterstützt. Der beobachtete Einfluss der zwei Adaptorproteine auf der Aktivierung von Ca2+-Kanälen in der Plasmamembran verschwindet, wenn die Speicher künstlich entleert sind. Daher scheinen die beiden Adaptorproteine ausschließlich die Ca2+-Mobilisierung aus intrazellulären Speichern zu beeinflussen. Obwohl beide Proteine einen ternären Komplex mit dem hemmenden Lipidphosphatase SHIP bilden, scheint diese Wechselwirkung für die Dok-3-Funktion nicht notwendig zu sein. Eher scheint SHIP die Dok-3-Phosphorylierung zu fördern. Koquervernetzung von Fc[gamma]RIIb mit dem BZR führt zu starker SHIP-Aktivierung und ich konnte zeigen, dass dies auch zu verstärkter Dok-3-Phosphorylierung führt. Diese Studie zeigt Einblicke in die molekularen Mechanismen, durch die die zytosolischen Adaptorproteine Dok-3 und Grb2 BZR-Signale regeln und identifiziert Fc[gamma]RIIb als Regulator des Dok-3-Grb2-Moduls.Signals emanating from the B cell antigen receptor (BCR) regulate both the activation of pathogen-specific B lymphocytes as well as removal of self-reactive B lymphocytes. The outcome of BCR stimulation can range from induction of apoptosis to proliferation. Inappropriate signals can therefore lead to an imbalance between humoral immunity and self tolerance. Consequently, B lymphocyte activation depends on a complex interplay between positive and negative signaling elements. The rise of the cytosolic Ca2+ concentration is a central step in B lymphocyte activation. The temporal shape of the Ca2+ signal contributes to the differential activation of transcription factors, which then affects cell fate decisions. The adaptor protein Grb2 has previously been shown to inhibit BCR-induced Ca2+ mobilization. During my work Björn Stork in our lab identified Dok-3 to be another adaptor protein inhibiting Ca2+ mobilization in B cells. In this study I investigated the molecular mechanisms of the inhibitory function of the two adaptor proteins. Using DT40 B cells that were rendered deficient for Grb2 or Dok-3 expression it could be shown that both proteins inhibit activation of the Ca2+ mobilizing enzyme PLC-[gamma]2 by Btk. This was evident from enhanced BCR-induced phosphorylation of PLC-[gamma]2 at a specific Btk phosphorylation site in the absence of Dok-3 or Grb2. This finding was supported by enhanced IP3 production and a stronger degree of depletion of Ca2+ from the ER stores in response to BCR ligation. An impact of the two adaptor proteins on the activation of plasma membrane Ca2+ channels could be excluded by uncoupling the release of Ca2+ from the ER from Ca2+ influx across the membrane. Although both proteins seem to form a ternary complex with the inhibitory lipid phosphatase SHIP, this interaction could be shown not to be essential for Dok-3 function. Rather, SHIP seems to promote Dok-3 phosphorylation. Fc[gamma]RIIb coligation to the BCR strongly activates SHIP and this could be shown to also enhance Dok-3 phosphorylation. Together, this study reveals insights in the molecular mechanism by which the cytosolic adaptor proteins Dok-3 and Grb2 regulate BCR signaling and identifies Fc[gamma]RIIb as an upstream regulator

C-reactive protein (CRP) recognizes uric acid crystals and recruits proteases C1 and MASP1

Gout is caused by crystallization of uric acid in the form of monosodium urate (MSU) crystals, which induce a sterile inflammatory response that is hardly distinguishable from microbe-induced inflammatory responses. It is unclear, if MSU crystals (like microbes) are recognized by specific pattern recognition receptors. To identify possible soluble pattern recognition molecules for MSU crystals, we purified MSU-binding proteins from human body fluids. We identified C-reactive protein (CRP) as a major MSU-binding protein. Binding of CRP was strong enough to specifically deplete CRP from human serum. We found that CRP was required for fixation of complement components C1q, C1r, C1s and MASP1. Thus, we have identified a pattern recognition molecule for MSU crystals that links to the activation of complement. Notably, CRP does not show an even binding to the complete surface of the crystals. It rather binds to edges or distinct faces of the crystals

Clec12a Is an Inhibitory Receptor for Uric Acid Crystals that Regulates Inflammation in Response to Cell Death

SummaryRecognition of cell death by the innate immune system triggers inflammatory responses. However, how these reactions are regulated is not well understood. Here, we identify the inhibitory C-type lectin receptor Clec12a as a specific receptor for dead cells. Both human and mouse Clec12a could physically sense uric acid crystals (monosodium urate, MSU), which are key danger signals for cell-death-induced immunity. Clec12a inhibited inflammatory responses to MSU in vitro, and Clec12a-deficient mice exhibited hyperinflammatory responses after being challenged with MSU or necrotic cells and after radiation-induced thymocyte killing in vivo. Thus, we identified a negative regulatory MSU receptor that controls noninfectious inflammation in response to cell death that has implications for autoimmunity and inflammatory disease

S100A9 is indispensable for survival of pneumococcal pneumonia in mice

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn$^{2+}$ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620)

Increased B-cell activity with consumption of activated monocytes in severe COVID-19 patients

The pathogenesis of autoimmune complications triggered by SARS-CoV2 has not been completely elucidated. Here, we performed an analysis of the cellular immune status, cell ratios, and monocyte populations of patients with COVID-19 treated in the intensive care unit (ICU) (cohort 1, N = 23) and normal care unit (NCU) (cohort 2, n = 10) compared with control groups: patients treated in ICU for noninfectious reasons (cohort 3, n = 30) and patients treated in NCU for infections other than COVID-19 (cohort 4, n = 21). Patients in cohort 1 presented significant differences in comparison with the other cohorts, including reduced frequencies of lymphocytes, reduced CD8+T-cell count, reduced percentage of activated and intermediate monocytes and an increased B/T8 cell ratio. Over time, patients in cohort 1 who died presented with lower counts of B, T, CD4+T, CD8+T-lymphocytes, NK cells, and activated monocytes. The B/T8 ratio was significantly lower in the group of survivors. In cohort 1, significantly higher levels of IgG1 and IgG3 were found, whereas cohort 3 presented higher levels of IgG3 compared to controls. Among many immune changes, an elevated B/T8-cell ratio and a reduced rate of activated monocytes were mainly observed in patients with severe COVID-19. Both parameters were associated with death in cohort 1

Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo

<p>Abstract</p> <p>Background</p> <p>Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs.</p> <p>Findings</p> <p>The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the <it>egfr </it>promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within <it>egfr </it>promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue.</p> <p>Conclusions</p> <p>The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme digestion enables a reliable and individual detection option for densely arranged binding motifs in vivo not provided by e.g. EMSA or in vivo footprinting. Given the important function of transcription factors in neoplastic mechanism, our method enables a broad diversity of application options for clinical studies.</p

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85

The Slp65 adaptor molecule is important for B-cell receptor signalling. Here, the interactomes of SLP65 in resting and activated B cells are resolved by mass spectrometry. A number of SLP65 interacting partners, including CIN85, continuously associate with SLP65 in a stimulation-independent manner. CIN85 recruits SLP65 to the membrane and it is required for SLP65 phosphorylation

Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane

<p>Abstract</p> <p>Background</p> <p>The chick chorio-allantoic membrane (CAM) assay is a commonly used method for studying angiogenic or anti-angiogenic activities <it>in vivo</it>. The ease of access allows direct monitoring of tumour growth by biomicroscopy and the possibility to screen many samples in an inexpensive way. The CAM model provides a powerful tool to study effects of molecules, which interfere with physiological angiogenesis, or experimental tumours derived from cancer cell lines. We therefore screened eight osteosarcoma cell lines for their ability to form vascularized tumours on the CAM.</p> <p>Findings</p> <p>We implanted 3-5 million cells of human osteosarcoma lines (HOS, MG63, MNNG-HOS, OST, SAOS, SJSA1, U2OS, ZK58) on the CAM at day 10 of embryonic development. Tumour growth was monitored by <it>in vivo </it>biomicroscopy at different time points and tumours were fixed in paraformaldehyde seven days after cell grafting. The tissue was observed, photographed and selected cases were further analyzed using standard histology.</p> <p>From the eight cell lines the MNNG-HOS, U2OS and SAOS were able to form solid tumours when grafted on the CAM. The MNNG-HOS tumours showed the most reliable and consistent growth and were able to penetrate the chorionic epithelium, grow in the CAM stroma and induce a strong angiogenic response.</p> <p>Conclusions</p> <p>Our results show that the CAM assay is a useful tool for studying osteosarcoma growth. The model provides an excellent alternative to current rodent models and could serve as a preclinical screening assay for anticancer molecules. It might increase the speed and efficacy of the development of new drugs for the treatment of osteosarcoma.</p