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(Investigative Ophthalmology and Visual Science. 2004;45:807-812.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0256
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Monoclonal Antibody (3G5)–Defined Ganglioside: Cell Surface Marker of Corneal Keratocytes

Brian M. Stramer,1,2,3 Michael G. K. Kwok,2 Pamela J. Farthing-Nayak,1,2,3,4 Jae-Chang Jung,1,2,6 M. Elizabeth Fini,1,2,3,5 and Ramesh C. Nayak1,2,4

1From the New England Eye Center, Tufts-New England Medical Center, Boston, Massachusetts; the 4Department of Ophthalmology, University of Arizona College of Medicine, Tucson, Arizona; the 2Departments of Ophthalmology and 3Anatomy and Cell Biology, Tufts University School of Medicine, Tufts Center for Vision Research, Boston, Massachusetts; the 5McKnight Vision Research Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida; and the 6Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, Korea.

PURPOSE. To evaluate the anti-ganglioside monoclonal antibody 3G5 as a marker of corneal keratocytes.

METHODS. 3G5 expression on keratocytes was investigated by immunofluorescence microscopy. Studies were performed on frozen sections of normal human, bovine, porcine, rabbit, rat, and mouse corneas and on repairing rabbit cornea. In vitro studies were performed on tissue-cultured human, bovine, porcine, mouse, and rabbit keratocytes.

RESULTS. 3G5 stained frozen sections of human, bovine, porcine, rat, and rabbit cornea but not mouse cornea and the staining pattern followed the distribution of stromal keratocytes but did not stain epithelium or endothelium. Subconfluent human and bovine keratocyte cultures were 3G5 negative. Almost 100% of the human and bovine cells that were maintained at confluence without replacement of serum-containing culture medium for 2 weeks became 3G5 positive. The 3G5 antigen was constitutively expressed on cultured rabbit and porcine keratocytes under all conditions examined. Mouse keratocyte cultures did not express 3G5. The 3G5 antigen was not present on myofibroblastic cells in the repairing area of a full-thickness wound in rabbit cornea that had been healing for 20 days. The area surrounding the healing wound expressed 3G5 antigen in an altered distribution, whereas 3G5 antigen was distributed in the expected pattern in areas that were distant from the wound. When rabbit keratocytes were induced to express the myofibroblast marker {alpha}-smooth muscle actin by treatment with TGFß1 in vitro, the pattern of 3G5 staining was altered.

CONCLUSIONS. The 3G5 antigen is a useful marker for the identification of corneal keratocytes and for documenting their response to environmental stimuli associated with wound repair.






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