Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cisand trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals.Cyclosporin A (CsA) and the structurally unrelated immunosuppressant FK506 have been demonstrated to inhibit T-cell activation and to be effective in organ transplantation (1-3). CsA has been shown to bind to and inhibit the rotamase activity of a cytoplasmic enzyme, cyclophilin (CyP) (4-6). Recently, a cytoplasmic receptor for FK506, FK506 binding protein (FKBP), which is unrelated in sequence to CyP, has also been shown to have similar rotamase activity, suggesting that this common enzymatic property of immunophilin proteins (immunosuppressant binding proteins) may be related to the regulation of T-cell activation (7-10). Rapamycin is a macrolide similar in structure to FK506 and, like FK506,12) MATERIALS AND METHODSReagents, Cell Culture, Interleukin 2 (IL-2) Production, and DNA Fragmentation. The IL-2-producing murine T-cell hybridoma 16.CD2-15.20 (15) was cultured (106 cells per well) with or without a 2% (vol/vol) culture supernatant of the anti-murine CD3 monoclonal antibody (mAb) 145-2C11 (16) in the absence or presence of CsA, FK506, or rapamycin. At 24 hr, culture supernatants were dialyzed and assayed for the presence of IL-2 as described (15). DNA was isolated from the cells and assayed for induction of DNA fragmentation or apoptosis on an ethidium bromide-stained...

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Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF‐AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c‐fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.

Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-KB, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate 13-galactosidase (13-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of 13-gal expression in which some cells express no 13-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which 13-gal activity is correlated with NF-ATbinding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-KB or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.

Exercise-induced bronchoconstriction (EIB) is a specific sign of active asthma, but its assessment in young children may be difficult with lung function techniques requiring active cooperation. The aim of the study was to assess the normal pattern of exercise-induced responses of respiratory impedance by using impulse oscillometry (IOS), and to investigate how these responses discriminate wheezy children from control subjects. IOS measurements were performed in a consecutive sample of wheezy children aged 3-7 years (n = 130) and in an aged matched control group of nonatopic children without respiratory symptoms (n = 79) before and after a free running test. After exercise, wheezy children showed significantly larger responses in respiratory resistance (Rrs5), reactance (Xrs5), and the resonance frequency (Fr) than the control subjects. In the control group, the upper 95% confidence limit of the maximal change was 32.5% for Rrs5, 85.7% for Xrs5, and 53.1% for Fr. By using analysis of receiver operating characteristics, the change in Rrs5 distinguished the wheezy children from the control subjects more effectively than change in Xrs5 or Fr. In wheezy children, the response was significantly effected by the outdoor temperature and exercise intensity in terms of maximum heart rate. In conclusion, an increase of 35% in Rrs5 after a free running test can be regarded as an abnormal response. Wheezy children show an enhanced airway response, which is clearly distinguishable from the control subjects. IOS is a feasible method to detect EIB in young children.