Article

Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, Good Samaritan Hospital 21239.
The Journal of Immunology (Impact Factor: 5.36). 12/1987; 139(9):3062-9.
Source: PubMed

ABSTRACT Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.

0 Bookmarks
 · 
65 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.
    Fish &amp Shellfish Immunology 07/2014; DOI:10.1016/j.fsi.2014.07.007 · 2.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The expression of FcR by human skin-derived mast cells of the MCTC type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of -hexosaminidase, PGD2, leukotriene C4 (LTC4), IL-5, IL-6, IL-13, GM-CSF, and TNF- .F cRIIa was consistently detected along with FcRI at the mRNA and protein levels; FcRIIc was sometimes detected only by RT-PCR; but FcRIIb, FcRI, and FcRIII mRNA and protein were not detected. FcRIIa-specific mAb caused skin MCTC cells to degranulate and secrete PGD2, LTC4, GM-CSF, IL-5, IL-6, IL-13, and TNF- in a dose-dependent fashion. FcRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC4, which was not released by this agonist. Simultaneous but independent cross-linking of FcRI and FcRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MCTC cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals. The Journal of Immunology, 2006, 177: 694 -701.
    The Journal of Immunology 06/2006; 177(1). DOI:10.4049/jimmunol.177.1.694 · 5.36 Impact Factor