Two-dimensional analysis of human lymphocyte proteins: I. An assay for lymphocyte effectors

We describe an assay for lymphocyte effectors that is capable of establishing the existence of regulators of lymphocyte gene expression (including post-transcriptional control and protein processing) and has the ability to characterize the response at the molecular level. The hypothesis that circulating effectors substances excreted through the kidney can be actively present in human urine was tested with this assay. Thus, biologically active protein molecules in urine were detected at concentrations of less than 1 mg/L and over a wide range of dilutions. Activities were detected and quantitated by culturing human lymphocytes with human urinary proteins in the presence of [35S]methionine and subsequently analyzing the labeled lymphocyte proteins by two-dimensional gel electrophoresis. Thus, protein analysis by two-dimensional gels was used to indirectly detect changes produced in cultured lymphocytes after exposure to regulatory molecules. Proteins or sets of lymphocyte proteins appeared or disappeared after exposure to normal or pathological human urinary proteins. Normal human urinary proteins triggered the appearance of sets of proteins referred to by number as the "Urocon" proteins and suppressed the synthesis of protein sets referred to as "Urocof" proteins. In addition to the normal alterations described, urinary proteins from individuals with influenza or acute leukemia and after renal transplantation were capable of inducing unique alterations in lymphocyte patterns.