S K Pixley1, A I Farbman, B P Menco. 1. Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Ohio 45267-0521, USA. sarah.pixley@uc.edu
Abstract
BACKGROUND: The olfactory epithelial sustentacular cells may support the survival and function of olfactory receptor neurons, but few reagents are available to mark and manipulate such cells. METHODS: Novel nasal cell-specific monoclonal antibodies were generated using whole cultured rat olfactory mucosal cells as the antigenic stimuli. They were characterized by immunostaining at the light level in rat tissues and newborn rat olfactory cell cultures, and at the electron microscopic level in adult tissues using freeze-substitution, post-embedding staining. RESULTS: An IgMkappa monoclonal antibody designated 1F4 selectively labeled apical surfaces of the rat olfactory and respiratory epithelia in tissue sections and what appeared to be sustentacular cells in olfactory cell cultures. Using electron microscopy, 1F4 bound selectively to the microvilli of sustentacular cells and ductal cells of Bowman's glands in the olfactory epithelium, and to the microvilli and cilia of ciliated but not secretory cells in the respiratory epithelium. No staining was detected in olfactory receptor neurons, basal cells, or two types of microvilli-bearing cells that differed from sustentacular cells. A contrasting antibody, 2H4, bound to granules of secretory respiratory cells. Developmental expression of 1F4 binding began at E17 and increased at and after E18/E19. Bulbectomy did not alter 1F4 immunoreactivity. Cell culture studies found that the 1F4 epitope was external and insensitive to trypsin treatment, and that both 1F4 and 2H4 positive cells required contact with aggregated cells for survival up to fifteen days in vitro. CONCLUSIONS: The antibody 1F4 is a useful marker and potential manipulation reagent specific for sustentacular cells and their microvilli.
BACKGROUND: The olfactory epithelial sustentacular cells may support the survival and function of olfactory receptor neurons, but few reagents are available to mark and manipulate such cells. METHODS: Novel nasal cell-specific monoclonal antibodies were generated using whole cultured rat olfactory mucosal cells as the antigenic stimuli. They were characterized by immunostaining at the light level in rat tissues and newborn rat olfactory cell cultures, and at the electron microscopic level in adult tissues using freeze-substitution, post-embedding staining. RESULTS: An IgMkappa monoclonal antibody designated 1F4 selectively labeled apical surfaces of the rat olfactory and respiratory epithelia in tissue sections and what appeared to be sustentacular cells in olfactory cell cultures. Using electron microscopy, 1F4 bound selectively to the microvilli of sustentacular cells and ductal cells of Bowman's glands in the olfactory epithelium, and to the microvilli and cilia of ciliated but not secretory cells in the respiratory epithelium. No staining was detected in olfactory receptor neurons, basal cells, or two types of microvilli-bearing cells that differed from sustentacular cells. A contrasting antibody, 2H4, bound to granules of secretory respiratory cells. Developmental expression of 1F4 binding began at E17 and increased at and after E18/E19. Bulbectomy did not alter 1F4 immunoreactivity. Cell culture studies found that the 1F4 epitope was external and insensitive to trypsin treatment, and that both 1F4 and 2H4 positive cells required contact with aggregated cells for survival up to fifteen days in vitro. CONCLUSIONS: The antibody 1F4 is a useful marker and potential manipulation reagent specific for sustentacular cells and their microvilli.