Membrane spanning 4A gene family expression and function in human mast cells

Mast cells are major effector cells of allergic and inflammatory disease. Thus understanding mechanisms of mast cell biology are of great interest to cell biology research. This study aimed to identify the expression and function of a newly identified gene family, the MS4A family, in human mast cells. This study identified 8 gene variants expressed in mast cells including 2 novel variants. All of the expressed genes were cloned and GFP and adenoviral constructs were generated. Quantitative RT-PCR demonstrated that all expressed gene variants were differentially regulated by mast cell stimulation with IgE, IgE/anti-lgE and stem cell factor (SCF) suggesting roles in mast cell biology. Transfections demonstrated that most proteins were trafficked to the cytoplasmic membrane, but some were trafficked to the nuclear membrane. This intracellular localisation of the MS4A family may be critical for their function. This was exemplified in this study by the function of a novel truncation (MS4A2trunc) of MS4A 2 (the beta chain of FceRI). MS4A2trunc was trafficked to the nuclear membrane, whereas MS4A 2 was trafficked to the cytoplasmic membrane. Overexpression of MS4A 2 using adenoviral transduction had no apparent effect on mast cell survival or proliferation. However, overexpression of MS4A2trunc profoundly inhibited mast cell proliferation and induced apoptosis via G2 phase cell cycle arrest. The removal of SCF from mast cell cultures upregulated the expression of MS4A2trunc. In addition, the slowly replicating primary human lung mast cells, and the mast cell line LAD-2 expressed MS4A2trunc. However, I was unable to demonstrate expression of MS4A2trunc in the rapidly replicating c-KIT gain-of-function mutated mast cell line HMC-1. Thus loss of expression of MS4A2trunc may be an important step in the development of mast cell neoplasia. This study has identified an entirely novel function for the MS4A 2 gene and has opened numerous avenues for future research on the MS4A family

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6

Background: Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival. Methods: Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA. Results: IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells. Conclusion: These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-5

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-4

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p>roducts. The product sizes corresponded to the expected size for both IL-6 and β-actin

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6

Background: Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival. Methods: Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA. Results: IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells. Conclusion: These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-0

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p> rapidly following cytokine withdrawal and this cell death is attenuated with the addition of IgE. IgE-induced HLMC survival is eliminated with the addition of an anti-IL-6 antibody and unaffected with the addition of an anti-SCF antibody following 7 days of culture. IgG does not promote HLMC survival when compared to the sodium azide control. Data is presented as the mean ± SEM from 6 different donors performed in triplicate. Blocking experiments are the mean ± SEM from 3 donors. Data for the IgG control experiments are the mean ± SEM from 4 donors. Donors in are different to those in and

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-6

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p> rapidly following cytokine withdrawal and this cell death is attenuated with the addition of IgE. IgE-induced HLMC survival is eliminated with the addition of an anti-IL-6 antibody and unaffected with the addition of an anti-SCF antibody following 7 days of culture. IgG does not promote HLMC survival when compared to the sodium azide control. Data is presented as the mean ± SEM from 6 different donors performed in triplicate. Blocking experiments are the mean ± SEM from 3 donors. Data for the IgG control experiments are the mean ± SEM from 4 donors. Donors in are different to those in and

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-1

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p>al time RT-PCR and upregulation of expression was confirmed using three different algorithms for calculation of results

IgE alone promotes human lung mast cell survival through the autocrine production of IL-6-3

<p><b>Copyright information:</b></p><p>Taken from "IgE alone promotes human lung mast cell survival through the autocrine production of IL-6"</p><p>http://www.biomedcentral.com/1471-2172/9/2</p><p>BMC Immunology 2008;9():2-2.</p><p>Published online 23 Jan 2008</p><p>PMCID:PMC2257927.</p><p></p>o evidence of non-specific amplification or primer-dimerisation. The graph is from a single experiment and representative of all donors

DataSheet_1_The exon-skipping oligonucleotide, KitStop, depletes tissue-resident mast cells in vivo to ameliorate anaphylaxis.docx

IntroductionAnaphylaxis represents the most extreme and life-threatening form of allergic disease and is considered a medical emergency requiring immediate intervention. Additionally, some people with mastocytosis experience recurrent episodes of anaphylaxis during normal daily activities without exposure to known triggers. While acute therapy consists primarily of epinephrine and supportive care, chronic therapy relies mostly on desensitization and immunotherapy against the offending allergen, which is a time-consuming and sometimes unsuccessful process. These treatments also necessitate identification of the triggering allergen which is not always possible, and thus highlighting a need for alternative treatments for mast cell-mediated diseases.MethodsThe exon-skipping oligonucleotide KitStop was administered to mice intradermally, intraperitoneally, or systemically at a dose of 12.5 mg/kg. Local mast cell numbers were enumerated via peritoneal lavage or skin histology, and passive systemic anaphylaxis was induced to evaluate KitStop’s global systemic effect. A complete blood count and biochemistry panel were performed to assess the risk of acute toxicity following KitStop administration.ResultsHere, we report the use of an exon-skipping oligonucleotide, which we have previously termed KitStop, to safely reduce the severity and duration of the anaphylactic response via mast cell depopulation in tissues. KitStop administration results in the integration of a premature stop codon within the mRNA transcript of the KIT receptor—a receptor tyrosine kinase found primarily on mast cells and whose gain-of-function mutation can lead to systemic mastocytosis. Following either local or systemic KitStop treatment, mice had significantly reduced mast cell numbers in the skin and peritoneum. In addition, KitStop-treated mice experienced a significantly diminished anaphylactic response using a model of passive systemic anaphylaxis when compared with control mice.DiscussionKitStop treatment results in a significant reduction in systemic mast cell responses, thus offering the potential to serve as a powerful additional treatment modality for patients that suffer from anaphylaxis.</p