Transforming growth factor beta-1 and beta-2 in human tear fluid

Purpose. To evaluate human tear fluid for transforming growth factor beta isoforms 1 and 2 (TGF-beta 1 and TGF-beta 2). Methods. To accomplish this, human tears were evaluated for TGF-beta s by quantitative antibody sandwich ELISA (sELISA), mink lung epithelial cell (MLEC) growth inhibition bioassay and western blotting. Various physical and chemical treatments were used to activate TGF-beta in these assays. Results. TGF-beta s could not be detected in untreated or heated tears by sELISA; however, mean TGF-beta 1 concentrations of 38.5 ng/ml and TGF-beta 2 concentrations of 2.32 ng/ml were detected in acid-activated tears by sELISA. Furthermore, 10.54 ng/ml of TGF-beta 1 and 3.98 ng/ml of TGF-beta 2 were detected in tears treated with the mucolytic agent, acetylcysteine. Total TGF-beta bioactivity in human tears measured by the MLEC assay was found to be 13.04 ng/ml in untreated tears and 24.85 ng/ml in acid-activated tears. Approximately one-half TGF-beta in tear speci mens was biologically active (mean = 52%, range 39-71%). Total tear TGF-beta bioactivity could be completely neutralized by recombinant human TGF-beta 1 latency associated peptide (rh TGF-beta 1 LAP). Mean neutralization of tear TGF-beta bioactivity was 83% by TGF-beta 1-specific antisera, and was 13% by TGF-beta 2-specific antisera. Immunoreactive TGF-beta bands at approximately 12.5 and 95 kD were observed in immunoblots of reduced acidified tears. A high molecular weight (MW) TGF-beta band (>203 kD) was noted in untreated tears; however, this band disappeared following treatment with acetylcysteine. Conclusions. The results of these studies indicate that TGF-beta 1 and TGF-beta 2 are present in human tear fluid, and TGF-beta 1 is the predominant isoform. There appears to be factors in human tears capable of binding TGF-beta.