Inhibition of cytokine production and cytokine-stimulated T-cell activation by FK506 (tacrolimus)1.

Insofar as it exerted its immunosuppressive effect by inhibiting cytokine expression, we assessed the effect of FK506 (Tacrolimus) on cytokine-stimulated T-cell activation. Human T cells, treated with FK506, or controls were stimulated with the mitogens PHA + PMA, Con A, and the "CD3-bypass" stimulation regimen, PMA + ionomycin. T-cell proliferation was quantitated by measuring the uptake of tritiated thymidine, and mRNA expression was assessed by RT-PCR. FK506, in a concentration-dependent fashion, inhibited T-cell proliferation and steady-state mRNA expression of IL-2 and IL-7; half-maximal suppression was obtained at 10(-7) to 5 x 10(-8) M. We tested whether FK506 antiproliferative effect could be overcome with exogenously reconstituted rIL-2 and/or rIL-7. Neither rIL-2 nor rlL-7, individually in conjunction with suboptimal concentrations of PHA or Con A, or in combination without any costimulus, was capable of abrogating FK506 antiproliferative effect, indicating that FK506 also acted by inhibiting cytokine-stimulated T-cell activation. To confirm this, T cells were treated with FK506 and stimulated by rIL-2 and rIL-7, individually in conjunction with suboptimal concentration of PHA and Con A. In addition, T cells were stimulated with rIL-2 and rIL-7 without any costimuli. FK506 inhibited T-cell activation stimulated by rIL-2 and by rIL-7, individually and in combination. This confirms that, in exerting its antiproliferative effect, FK506 acts at two levels, by inhibiting cytokine availability and by suppressing cytokine effect on target cells, and explains the beneficial effect of FK506 in attenuating ongoing immune responses.

Structures of type 2 peroxisomal targeting signals in two trypanosomatid aldolases.

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Opposing effects of rapamycin and cyclosporin A on activation-induced Ca(2+) release.

Insofar as Ca(2+) plays a major role in T cell activation, we investigated the effect of the immunosuppressants cyclosporin A and rapamycin on T cell proliferation and on the activation-induced increase in [Ca(2+)](i). Both cyclosporin A and rapamycin inhibited mitogen (concanavalin A and phytohemagglutinin) and ionomycin+phorbol myristate acetate (PMA)-driven T cell proliferation (Ca(2+)-dependent). However, only rapamycin suppressed T cell proliferation stimulated by anti-CD28 antibody (Ab)+PMA, and recombinant interleukin-6-stimulated proliferation of the interleukin-6 dependent B9 cells (Ca(2+)-independent). These differences were associated with a different effect of both drugs on Ca(2+) release, as cyclosporin A attenuated while rapamycin augmented the mitogen-induced elevation in [Ca(2+)](i). Collectively, this supports the notion that Ca(2+) is required in early stages of T cell activation, and that cyclosporin A blocked only Ca(2+)-dependent while rapamycin blocked both Ca(2+)-dependent and -independent events of T cell activation.

Pretreatment with glucocorticoids enhances T-cell effector function: possible implication for immune rebound accompanying glucocorticoid withdrawal.

Glucocorticoids (GCs) exert their immunosuppressive/antiproliferative effects largely through inhibition of cytokine expression, and paradoxically upregulate the expression of (proinflammatory) cytokine receptors on select nonlymphoid cells. Clinically, withdrawal of GCs was frequently associated with worsening of the outcome of heightened immunity disorders, thereby implicating enhanced cytokine and cytokine receptor expression as a possible consequence of acute/short-term GCs withdrawal. In view of the significance of this complication of GC therapy, we addressed the effect of GC withdrawal on cytokine receptor expression and subsequent T-cell effector function, using the proliferation of human T cells as biological readout. To mimic GC withdrawal, T cells were treated with GCs or controls, stimulated, and incubated for 16-20 h at 37 degrees C, washed, and reactivated for a further 4-48 h. Surface marker expression was assessed by FACS analysis, and proliferation was determined by measuring the cellular uptake of tritiated thymidine. Dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent manner, inhibited T-cell proliferation induced by anti-CD28 Ab + PMA. However, pretreatment of T cells activated with mitogens, cross-linking antibodies, or PMA + ionomycin ("CD3-bypass" stimulation regimen), but not resting T cells, with DEX or PRED resulted in a marked increase in IL-IR, IL-2R alpha, and IL-6R expression, which was accompanied by a significant enhancement in T-cell proliferation. This effect of GCs was neither stimulus specific nor did it result from alteration in cell viability, and was paralleled by augmentation in cytokine (rIL-2) effects on DEX-pretreated and preactivated T cells. Taken together, our results underline the dual effects of GCs in regulating T-cell activation and cytokine expression. In essence, GCs directly inhibited T-cell proliferation by suppressing cytokine production, and, by enhancing cytokine receptor expression, pretreatment with GCs augmented T-cell proliferation.

In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.