Corneal endothelium modulation factor released by polymorphonuclear leukocytes. Partial purification and initial characterization

E. P. Kay, L. Rivela, Y. G. He

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Abstract

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first passaged cells contained type IV collagen RNA In an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.

Original languageEnglish (US)
Pages (from-to)313-322
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume31
Issue number2
StatePublished - 1990

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Corneal Endothelium
Neutrophils
Collagen Type IV
Endothelial Cells
Collagen
Collagen Type I
Cell Shape
Phenotype
DEAE-Dextran
Antibodies
Ammonium Sulfate
Trypsin
Chromatography
Complementary DNA
RNA
Staining and Labeling
Rabbits
Gene Expression
Messenger RNA
Peptides

Keywords

  • collagen gene expression
  • collagen MRNAs
  • corneal endothelial cells
  • modulation factor
  • polymorphonuclear leukocyte

ASJC Scopus subject areas

  • Ophthalmology

Cite this

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AU - Kay, E. P.

AU - Rivela, L.

AU - He, Y. G.

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N2 - Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first passaged cells contained type IV collagen RNA In an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.

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