Human mast cells (HMCs) are derived from a CD34+ pluripotent progenitor cell that is Kit (CD117+), CD13+, Fc epsilonRI- and lacks lineage-specific surface markers. Bone marrow and peripheral blood are two tissue sources available for obtaining CD34+ progenitor cells from which to culture HMCs. CD34+ cells can be isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. We show that enriched CD34+ cells may also be immediately cultured in serum-free culture media containing recombinant human stem cell factor (rhSCF), rhIL-6, and rhIL-3 (first week only). Weekly hemidepletions and the removal of adherent cells and/or debris enables the investigator to obtain HMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 wk.? Mast cells are involved in both the genesis of allergic inflammation and in host defense; and reside in tissues where their location and responsiveness is regulated in part by adhesion to extracellular matrix proteins (ECM). We have reported that human mast cells (huMC) express TLR1-7, and 9 and respond to toll-like receptors (TLR) ligands by releasing cytokines and leukotriene C4. To determine if TLR ligation could similarly affect mast cells via an influence on adhesion, we employed huMC; and as substrates, fibronectin (FN) and vitronectin (VN). huMC were thus treated with double-stranded RNA (dsRNA) and adhesion to ECM was quantified. FcvarepsilonRI dependent mast cell degranulation was assessed. Adhesion molecule expression and activation was measured by flow cytometry. Activation of huMC through TLR3 with increasing amounts of polyI:C inhibited mast cell adhesion in a dose-dependent manner. This decrease in adhesion was accompanied by a similar decrease in IgE-mediated mast cell degranulation. Activation of TLR3 on huMC resulted in a change in the conformation of CD29, the receptor for FN, to an inactive form. Thus, TLR3 activation decreases mast cell attachment to VN and FN through an active process and one, which would abrogate mast cell attachment dependent potentiation of IgE-mediated responses.? Mast cells, which are associated with T helper cell type 2-dependent inflammation, have now been implicated in the innate immune response. To further characterize how mast cells are programmed to respond to infectious organisms, we used expression profiling using DNA microarray analysis of gene expression by human mast cells (huMC) during ingestion of Escherichia coli and examined immunoglobulin E (IgE)-mediated degranulation. Analysis of data revealed that specific groups of genes were modulated, including genes encoding transcription factors, cell signaling molecules, cell cycle regulators, enzymes, cytokines, novel chemokines of the CC family, adhesion molecules, and costimulatory molecules. Enzyme-linked immunosorbent assay analysis confirmed the production of tumor necrosis factor and the chemokines CC chemokine ligand (CCL)-1/I-309, CCL-19/macrophage-inflammatory protein-3beta (MIP-3beta), and CCL-18/MIP-4; flow cytometry confirmed the up-regulation of carcinoembryonic antigen-related cell adhesion molecule 1, the integrin CD49d, and CD80. Coincubation with E. coli down-regulated Fc receptor for IgE I (FcepsilonRI) expression and FcepsilonRI-mediated huMC degranulation. These data are consistent with the concept that bacterial exposure directs mast cell responses toward innate immunity and away from IgE-mediated effects.
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| Sundstrom, J Bruce; Ellis, Jane E; Hair, Gregory A et al. (2007) Human tissue mast cells are an inducible reservoir of persistent HIV infection. Blood 109:5293-300 |
| Zudaire, Enrique; Martinez, Alfredo; Garayoa, Mercedes et al. (2006) Adrenomedullin is a cross-talk molecule that regulates tumor and mast cell function during human carcinogenesis. Am J Pathol 168:280-91 |
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