SummaryThe regulation of mKNA encoding transforming growth factor 3 (TGF-/3) and interleukin 2 (I1,2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-3 mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-l,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-B mRNA in the stimulated T cells, in contrast to that of II.2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-3 mRNA levels is also appreciable at the level of production of functionally active TGF-3 protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.T ransforming growth factor 3 (TGF-3), a 25-kD homodimeric multifunctional cytokine and a secretory product of many cell types including T cells, is a potent inhibitor of T cell growth and differentiation (1-3). I1,2, on the other hand, promotes T cell growth and their acquisition of specialized effector functions (4). TGF-B and I1,2, thus, have the potential to exert diametrically opposite effects on the expression of antigen-specific T cell immunity. It was of interest, therefore, to quantify, simultaneously, the regulation of mRNA encoding TGF-3 and Ib2 in normal human T cells. Also, in view of the ability of TGF-3 to inhibit the antiallograft response (5), it was considered important to quantify the effect of cyclosporine (CsA) on the induction of TGF-fl mKNA in normal human T cells, and compare it to its well-characterized inhibitory effect on the induction of II.-2 mRNA (6). Materials and Methods Isolation and Activation of T Cells. T cells were isolated fromnormal human PBMC with a sequential muhi-step procedure that yields >98% CD2 antigen-positive cells and <1% cells that are positive for the DR, CD14, CD25, or CD56 antigens (7). Accessory cell-independent T cell activation was accomplished with either sn-l,2-dioctanoylglycerol (DAG; 10.0/zg/ml) and ionomycin (1.0 #M) or crosslinked anti-CD2 (OKT11; 0.5 #g/ml) and anti-CD3 (OKT3; 0.5 #g/ml) mAbs (7).Design and Synthesis of Competitor DNA Constructs. Fig. 1 illus-trates the design, synthesis, and authentication of the 290-bp TGF-/~ competitor DNA construct created for the quantification of TGF-B mRNA by PCR. As shown, the oligonucleotide primer pair was designed to amplify a region in the TGF-3 gene that contains a MseI restriction site. The MseI digestion of the 246-bp TGF-3 PCK product yielded 210-and 36-bp subfragments that were annealed with a 44-bp DNA insert synthesized in vitro to have cohesive en...

Hypertension, a remediable risk factor for stroke, cardiovascular disease, and renal failure, affects 50 million individuals in the United States alone. African Americans (blacks) have a higher incidence and prevalence of hypertension and hypertension-associated target organ damage compared with Caucasian Americans (whites). Herein, we explored the hypotheses that transforming growth factor-beta(1) (TGF-beta(1)) is hyperexpressed in hypertensives compared with normotensives and that TGF-beta(1) overexpression is more frequent in blacks compared with whites. These hypotheses were stimulated by our recent demonstration that TGF-beta(1) is hyperexpressed in blacks with end-stage renal disease compared with white end-stage renal disease patients and by the biological attributes of TGF-beta(1), which include induction of endothelin-1 expression, stimulation of renin release, and promotion of vascular and renal disease when TGF-beta(1) is produced in excess. TGF-beta(1) profiles were determined in black and white hypertensive subjects and normotensive controls and included circulating protein concentrations, mRNA steady-state levels, and codon 10 genotype. Our investigation demonstrated that TGF-beta(1) protein levels are highest in black hypertensives, and TGF-beta(1) protein as well as TGF-beta(1) mRNA levels are higher in hypertensives compared with normotensives. The proline allele at codon 10 (Pro(10)) was more frequent in blacks compared with whites, and its presence was associated with higher levels of TGF-beta(1) mRNA and protein. Our findings support the idea that TGF-beta(1) hyperexpression is a risk factor for hypertension and hypertensive complications and provides a mechanism for the excess burden of hypertension in blacks.