PMID- 9477991
OWN - NLM
STAT- MEDLINE
DCOM- 19980302
LR  - 20131121
IS  - 0146-0404 (Print)
IS  - 0146-0404 (Linking)
VI  - 39
IP  - 2
DP  - 1998 Feb
TI  - 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.
PG  - 336-43
AB  - 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.
FAU - Cook, E B
AU  - Cook EB
AD  - Department of Medicine, School of Medicine, University of Wisconsin-Madison 
      53792, USA.
FAU - Stahl, J L
AU  - Stahl JL
FAU - Miller, S T
AU  - Miller ST
FAU - Gern, J E
AU  - Gern JE
FAU - Sukow, K A
AU  - Sukow KA
FAU - Graziano, F M
AU  - Graziano FM
FAU - Barney, N P
AU  - Barney NP
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Invest Ophthalmol Vis Sci
JT  - Investigative ophthalmology & visual science
JID - 7703701
RN  - 0 (DNA Primers)
RN  - 0 (Ionophores)
RN  - 0 (RNA, Messenger)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 126547-89-5 (Intercellular Adhesion Molecule-1)
RN  - 37H9VM9WZL (Calcimycin)
RN  - EC 3.4.21.- (Serine Endopeptidases)
RN  - EC 3.4.21.- (chymase 2)
RN  - EC 3.4.21.39 (Chymases)
RN  - EC 3.4.21.59 (Tryptases)
SB  - IM
MH  - Calcimycin/pharmacology
MH  - Cell Separation
MH  - Cells, Cultured
MH  - Chymases
MH  - Conjunctiva/*cytology
MH  - DNA Primers/chemistry
MH  - Epithelial Cells/drug effects/*metabolism
MH  - Flow Cytometry
MH  - Humans
MH  - Intercellular Adhesion Molecule-1/genetics/*metabolism
MH  - Ionophores/pharmacology
MH  - Mast Cells/chemistry/*physiology/ultrastructure
MH  - Polymerase Chain Reaction
MH  - RNA, Messenger/metabolism
MH  - Recombinant Proteins/pharmacology
MH  - Serine Endopeptidases/metabolism
MH  - Transcription, Genetic
MH  - Tryptases
MH  - Tumor Necrosis Factor-alpha/pharmacology/*physiology
MH  - Up-Regulation
EDAT- 1998/03/07 00:00
MHDA- 1998/03/07 00:01
CRDT- 1998/03/07 00:00
PHST- 1998/03/07 00:00 [pubmed]
PHST- 1998/03/07 00:01 [medline]
PHST- 1998/03/07 00:00 [entrez]
PST - ppublish
SO  - Invest Ophthalmol Vis Sci. 1998 Feb;39(2):336-43.