February 1998
Volume 39, Issue 2
Free
Articles  |   February 1998
Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells.
Author Affiliations
  • E B Cook
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • J L Stahl
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • S T Miller
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • J E Gern
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • K A Sukow
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • F M Graziano
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
  • N P Barney
    Department of Medicine, School of Medicine, University of Wisconsin-Madison 53792, USA.
Investigative Ophthalmology & Visual Science February 1998, Vol.39, 336-343. doi:
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      E B Cook, J L Stahl, S T Miller, J E Gern, K A Sukow, F M Graziano, N P Barney; Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells.. Invest. Ophthalmol. Vis. Sci. 1998;39(2):336-343.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

PURPOSE: To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro. METHODS: Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient. Epithelial cells obtained from the top layer of the gradient were cultured to confluence. Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient. Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message. Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein. An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis. RESULTS: Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue. RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA. An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha. ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor. This upregulation was blocked by incubation with TNF alpha-neutralizing antibody. CONCLUSIONS: This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.

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