Marrow-derived Mouse Mast Cells Research Articles

Agarose/crystalline nanocellulose (CNC) composites promote bone marrow-derived mast cell integrity, degranulation and receptor expression but inhibit production of de novo synthesized mediators.

Introduction: Mast cells are highly granulated tissue-resident leukocytes that require a three-dimensional matrix to differentiate and mediate immune responses. However, almost all cultured mast cells rely on two-dimensional suspension or adherent cell culture systems, which do not adequately reflect the complex structure that these cells require for optimal function. Methods: Crystalline nanocellulose (CNC), consisting of rod-like crystals 4-15nm in diameter and 0.2-1µm in length, were dispersed in an agarose matrix (12.5% w/v), and bone marrow derived mouse mast cells (BMMC) were cultured on the agarose/CNC composite. BMMC were activated with the calcium ionophore A23187 or immunoglobulin E (IgE) and antigen (Ag) to crosslink high affinity IgE receptors (FcεRI). Results: BMMC cultured on a CNC/agarose matrix remained viable and metabolically active as measured by reduction of sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the cells maintained their membrane integrity as analyzed by measuring the release of lactate dehydrogenase (LDH) and propidium iodide exclusion by flow cytometry. Culture on CNC/agarose matrix had no effect on BMMC degranulation in response to IgE/Ag or A23187. However, culture of BMMC on a CNC/agarose matrix inhibited A23187-and IgE/Ag-activated production of tumor necrosis factor (TNF) and other mediators such as IL-1β, IL-4, IL-6, IL-13, MCP-1/CCL2, MMP-9 and RANTES by as much as 95%. RNAseq analysis indicated that BMMC expressed a unique and balanced transcriptome when cultured on CNC/agarose. Discussion: These data demonstrate that culture of BMMCs on a CNC/agarose matrix promotes cell integrity, maintains expression of surface biomarkers such as FcεRI and KIT and preserves the ability of BMMC to release pre-stored mediators in response to IgE/Ag and A23187. However, culture of BMMC on CNC/agarose matrix inhibits BMMC production of de novo synthesized mediators, suggesting that CNC may be altering specific phenotypic characteristics of these cells that are associated with late phase inflammatory responses.

A novel role for the adapter molecule NHERF1 in regulating mast cell-mediated anaphylaxis

Abstract Anaphylaxis is a life threatening allergic reaction that is classically elicited by crosslinking of antigen-specific IgE bound to the high affinity IgE receptor FcɛRI on mast cells. Proteases released from FcɛRI-activated mast cells result in the accumulation of the anaphylatoxin, C3a that can further amplify the anaphylactic response via interaction with C3a receptor (C3aR), a G-protein coupled receptor (GPCR). This autocrine loop resulting in C3aR activation as a consequence of FcɛRI stimulation initiates anaphylaxis in humans. C3aR possess a class I PDZ binding motif on its cytoplasmic tail. Adapter proteins such as the Na+/H+ exchanger regulatory factor 1 (NHERF1) have been implicated in regulating GPCR functions by binding to the class I PDZ motifs in these receptors. In our previous study, we had demonstrated that NHERF1 promotes C3a-induced degranulation and chemokine production in human mast cells. The aim of this study was to determine if NHERF1 regulates mouse mast cell response following FcɛRI stimulation. Our data suggests that NHERF1 enhances intracellular Ca2+ mobilization, activation of the MAP kinase pathway (ERK and P38) and production of cytokines (IL-6 and IL-13) following IgE/antigen-mediated activation in bone marrow derived mouse mast cells. In agreement with our in vitro data, NHERF1 promotes mast cell-mediated passive systemic anaphylaxis and passive cutaneous anaphylaxis induced by IgE and antigen in clinically relevant murine models. These results thus indicate that the NHERF1 plays a vital role in regulating mast cell-mediated anaphylaxis and that NHERF1 could be a potential novel target for anaphylaxis and other allergic diseases.

Intracellular IL-15 controls mast cell survival

The regulation of mast cell activities and survival is a central issue in inflammatory immune responses. Here, we have investigated the role of mouse interleukin-15, a pro-inflammatory and pleiotropic cytokine, in the control of mast cell survival and homeostasis. We report that aged IL-15−/− mice show a reduced number of peritoneal mast cells compared to WT mice. Furthermore, IL-15 deficiency in bone marrow derived mouse mast cells (BMMCs) results in increased susceptibility to apoptosis mediated by growth factor deprivation and A-SMase-treatment. IL-15−/− BMMCs show a constitutive stronger mRNA and protein expression as well as enzymatic activity of the members of the mitochondrial apoptotic pathways including acidic lysosomal aspartate protease cathepsin D (CTSD), endogenous acid sphingomyelinase (A-SMase), caspase-3 and -7 compared to wild type (WT) BMMCs. Furthermore, IL-15−/− BMMCs constitutively generate more A-SMase-derived ceramide than WT controls and display a decreased expression of pro-survival sphingosin-1-phosphate (SPP) both in cytosol and membrane cell fractions. Furthermore, pre-treatment of mast cells with imipramine or pepstatin A, inhibitors of the intracellular acid sphingomyelinase and cathepsin D pathways respectively, increases survival in IL-15−/− BMMCs. These findings suggest that intracellular IL-15 is a key regulator of pathways controlling primary mouse mast cell homeostasis.