Erythromycin inhibits transcriptional activation of NF-kappaB, but not NFAT, through calcineurin-independent signaling in T cells

Abstract

The molecular mechanism of the anti-inflammatory effect of erythromycin (EM) was investigated at the level of transcriptional regulation of cytokine gene expression in T cells. EM (>10(-6) M) significantly inhibited interleukin-8 (IL-8) expression but not IL-2 expression from T cells induced with 20 ng of phorbol 12-myristate 13-acetate (PMA) per ml plus 2 microM calcium ionophore (P-I). In electrophoretic mobility shift assays EM at 10(-7) to 10(-5) M concentrations inhibited nuclear factor kappa B (NF-kappaB) DNA-binding activities induced by P-I. Reporter gene assays also showed that EM (10(-5) M) inhibited IL-8 NF-kappaB transcription by 37%. The inhibitory effects of EM on transcriptional activation of IL-2 and DNA-binding activity of nuclear factor of activated T cells (NFAT) were not seen in T cells. On the other hand, FK506, which is also a macrolide derivative, inhibited transcriptional activation of both NF-kappaB and NFAT more strongly than EM did. The mechanism of EM inhibition of transactivation of NF-kappaB was further investigated in transiently transfected T cells that express calcineurin A and B subunits. Expression of calcineurin did not render transactivation of NF-kappaB in T cells more resistant to EM, while the inhibitory effect of FK506 on transactivation of NF-kappaB was attenuated. These findings indicate that EM is capable of inhibiting expression of the IL-8 gene in T cells through transcriptional inhibition and that this inhibition is mediated through a non-calcineurin-dependent signaling event in T lymphocytes.

FIG. 1

Inhibitory effects of EM and FK506 on transcriptional activation of IL-8 (a) or IL-2 (b) gene expression. Transgenic Jurkat T-cell lines that stably express the firefly luciferase gene driven by full-length human IL-8 promoter (positions −1451 to +44) or human IL-2 enhancer (positions −326 to +45) were established (see Materials and Methods). These T-cell lines were pretreated with either EM or FK506 at a dose range of 10⁻⁹ to 10⁻⁴ M for 1 h and were then stimulated with PMA (20 ng/ml) plus ionomycin (2 μM) for 4 h in the presence of the drugs. Ten micrograms of whole-cell extracts was used for the luciferase assay. Transcriptional activation is shown as the enzymatic activities of luciferase contained in whole cells. Data represent means ± standard deviations for three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 2

Inhibitory effects of EM and FK506 on transcriptional activation of cis-acting enhancer elements. Transgenic Jurkat T cells that stably express the firefly luciferase gene driven by IL-8 NF-κB (positions −84 to +44) (a) or three tandem copies of IL-2 NFAT (b) were established (see Materials and Methods). These T-cell lines were pretreated with either EM or FK506 at a dose range of 10⁻⁹ to 10⁻⁴ M for 1 h and were then stimulated with PMA (20 ng/ml) plus ionomycin (2 μM) for 4 h in the presence of the drugs. The cells were then subjected to the luciferase assay as described in the legend to Fig. 1.

FIG. 3

Induction and drug modulation of DNA-binding activities of nuclear transcriptional proteins NF-κB and NFAT in plain Jurkat T cells. The cells were either nonstimulated (NS) or stimulated with PMA (20 ng/ml) or PMA plus ionomycin (2 μM) (PMA/Iono) for 2 h. Inhibition by EM (10⁻⁷ to 10⁻⁵ M) or FK506 (10⁻⁶ M) of P-I-induced specific DNA-binding activities was also tested. Ten micrograms of NEs was incubated with the ³²P-labeled consensus sequence of IL-8 NF-κB (a) or IL-2 NFAT (b) oligonucleotides in the presence of 1 μg of poly(dI-dC), and the NE-DNA complexes were resolved by EMSAs.

FIG. 4

Involvement of calcineurin in transcriptional activation of IL-8 NF-κB. (a) Attenuation of IL-8 NF-κB sensitivity to FK506 inhibition by calcineurin expression in plain Jurkat T cells. All transfections include the IL-8 NF-κB luciferase reporter plasmid and either the pEF/CaA-expressing catalytic subunit of calcineurin (closed squares), the pEF/CaA- plus pEF/CaB-expressing regulatory subunits of calcineurin (open squares), or mock plasmid pEF(−) (open circles). At 15 h following transient cotransfection, the cells which had already been treated for 1 h with FK506 were stimulated with PMA (20 ng/ml) plus ionomycin (2 μM) (PMA/Iono) for 4 h in the presence of the drug. (b) Upregulation of transcriptional activation of IL-8 NF-κB by constitutively active calcineurin in an FK506-sensitive manner. Plain Jurkat T cells were transiently cotransfected with reporter plasmid pκB-8/luc and either a control expression vector, pEF(−) (Control) or pEF/ΔCaM-AI, that constitutively expresses the catalytic subunit of calcineurin (ΔCaM-AI). Following 15 h of recovery, the cells were pretreated with FK506 and were then stimulated as described above for panel a. Whole-cell extracts were prepared, and luciferase assays were performed as described in Materials and Methods. NS, nonstimulated; FK, FK506. In each experiment, the luciferase activity produced was normalized to the amount of β-galactosidase activity and protein concentrations. Data represent means ± standard deviations for three independent experiments.

FIG. 5

Effects of calcineurin expression on sensitivity of IL-8 NF-κB transactivation by EM in T cells. All transfections include the IL-8 NF-κB luciferase reporter plasmid and either pEF/CaA plus pEF/CaB (closed squares) or the control expression vector pEF(−) (open squares). Following 15 h of incubation, Jurkat T cells were pretreated for 1 h with various concentrations of EM and were then stimulated with PMA (20 ng/ml) plus ionomycin (2 μM) for 4 h in the presence of EM. Luciferase activities were analyzed as described in Materials and Methods. Data represent means ± standard deviations for three independent experiments.