Studies on Ciliary Dyskinesia Factor in Cystic Fibrosis. III. Skin Fibroblasts and Cultured Amniotic Fluid Cells

Abstract

Extract: The cell-free media from cultured skin fibroblasts derived from 16 healthy subjects, 13 homozygotes, 6 obligate heterozygotes, and 1 potential heterozygote for cystic fibrosis (CF) were investigated for ciliary dyskinesia factor (CDF) by a modified rabbit tracheal bioassay. Fluid was obtained for study from cultures during growth phase, 24 and 48 hr after feeding, before confluence had been reached. A positive CDF response was demonstrated in the media of all homozygous and heterozygous for CF skin fibro-blast cultures, after incubation of the culture fluid with human immunoglobulin G (IgG). No CDF response was observed before the addition of IgG. Although there was a tendency of the cell-free media obtained from the CF homozygous fibroblast cultures to elicit a stronger CDF response than the media from heterozygous cultures, no consistent distinction between homozygous and heterozygous fibroblast cultures could be obtained. The fluid of 15 out of 16 fibroblast cultures derived from normal donors did not demonstrate CDF activity before or after the addition of IgG. The media from cultured amniotic fluid cells obtained from a pregnant woman homozygous for CF and from four pregnancies derived from heterozygous parents demonstrated CDF activity in the presence of IgG. The media of 10 out of 11 control amniotic fluid cell cultures were CDF negative. In the one CDF-positive control culture a positive CDF response was also elicited in the maternal serum. Media from CF fibroblast cultures, to which IgG had been added, lost their CDF activity after treatment with anti-IgG. The culture fluid from confluent fibroblast and amniotic fluid cell cultures of all types of subjects, including control subjects, inhibited the ciliary activity of rabbit tracheal explants without addition of IgG. No CDF activity was observed in the amniotic fluid of a fetus affected with CF. Amicon filtration experiments demonstrated that a molecule with molecular weight between 1,000 and 10,000 produced by CF fibroblast cultures causes ciliary dyskinesia in the presence of IgG. The findings suggest that cultured skin fibroblasts and amniotic fluid cells that carry the CF gene in a homozygous or heterozygous state produce a small molecule, preciliary dyskinesia factor (pre-CDF), which is activated in the presence of IgG, as tested with the rabbit bioassay. It appears that this activation of pre-CDF could be reversed by its removal from the IgG. Speculation: Cells from all individuals produce a pre-CDF, which is a normal product important for rapid changes in electrolyte flux at the cell membrane. This molecule is normally rapidly inactivated by an appropriate enzyme. The genetic defect in CF is in the reduced activity of this enzyme, which results in abnormalities at the cell membrane because of excess of pre-GDF, and, after activation of pre-CDF to CDF by IgG, in interference with bronchial ciliary function.