Glucocorticoids attenuate T cell receptor signaling

Abstract

Glucocorticoids (GCs) affect peripheral immune responses by inhibiting T cell immunity at several stages of the activation cascade, causing impaired cytokine production and effector function. The recent demonstration that the thymic epithelium and possibly thymocytes themselves produce steroids suggests that endogenous GCs also play a role in the control of T cell development. As both peripheral responsiveness and thymic differentiation appear to be regulated by the quantity and quality of intracellular signals issued by antigen-major histocompatibility complex-engaged T cell receptor (TCR) complexes, we investigated the effects of GCs on the signaling properties of T cells stimulated by anti-CD3 monoclonal antibodies or agonist peptides. We demonstrate in this work that dexamethasone, a synthetic GC, inhibits the early signaling events initiated upon TCR ligation, such as tyrosine phosphorylation of several TCR-associated substrates including the zeta chain, the ZAP70 kinase, and the transmembrane adapter molecule linker for activation of T cells. Hypophosphorylation was not a consequence of reduced kinase activity of src protein tyrosine kinases, but was correlated with an altered- membrane compartmentalization of these molecules. These observations indicate that in addition to their well-described ability to interfere with the transcription of molecules involved in peripheral responses, GCs inhibit T cell activation by affecting the early phosphorylating events induced after TCR ligation.

Figure 1

DEX causes partial signaling in anti-CD3–stimulated T cell hybridoma cells. (A) T cell hybrids (3 × 10⁴ cells/well) were stimulated by graded doses of plastic-adsorbed anti-CD3ε mAbs in control or 1 μM DEX-supplemented media. Supernatants were collected after overnight culture and IL-2 content was determined using a standard ELISA. Results are expressed as the mean ± SD of triplicate determinations. TCR signaling studies were performed as follows: T cell hybridomas (2 × 10⁷ cells/lane) were cultured for 16 h with 1 μM DEX or solvent (ethanol). An equivalent number of cells were left untreated or stimulated by TCR cross-linking for 2 min and lysed in Brij97 lysis buffer. (B) Lysates were immunoprecipitated (IP) with Sepharose beads coupled to anti-CD3ε or anti-CD3ζ mAbs as indicated, resolved by SDS-PAGE, and immunoblotted (IB) with antiphosphotyrosine mAbs. The blot was subsequently stripped and reprobed with an mAb against CD3ζ and a ZAP70-specific rabbit antiserum. (C) The intensity of phospho-ζ isoforms resolved by SDS-PAGE from nine independent experiments was quantitated by densitometric analysis. Results are expressed as percent inhibition of the relative intensity of bands in DEX- versus control-treated cells (mean ± SD). (D and E) Lysates were immunoprecipitated with rabbit antisera raised against ZAP70 and LAT. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine mAbs. Stripped membranes were reprobed with antisera raised against ZAP70 and LAT (bottom). Similar results were obtained in three independent experiments. D, DEX.

Figure 2

Inhibition of TCR signaling by DEX requires binding to the GC receptor and de novo protein synthesis. (A) T cell hybridomas were incubated with media or 1 μM DEX for the indicated times before stimulation by anti-CD3 mAbs. T cell hybridomas were preincubated for 2 h in the presence of (B) the GC receptor antagonist RU486 (1 μM RU) or (C) the protein synthesis inhibitor cycloheximide (0.25 μg/ml CHX) before addition of 0.1 μM DEX. The signaling properties of these cells were analyzed after a 16-h incubation period as described in the legend for Fig. 1. The results are representative of three independent experiments. D, DEX. IP, immunoprecipitation.

Figure 3

DEX inhibits the early steps of TCR signaling in thymocytes. (A) Balb/c thymocytes were incubated for 16 h in the indicated concentrations of DEX or solvent (ethanol). Cultured thymocytes were left untreated or stimulated by anti-CD3 mAbs at 37°C for 2 min and lysed in Brij97 lysis buffer. Cell lysates (2 × 10⁷ cells/lane) were immunoprecipitated (IP) with an anti-CD3ε mAb directly coupled to beads. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted (IB) with antiphosphotyrosine mAbs. The amount of protein was compared by reblotting the membrane with anti-CD3ζ mAbs (bottom). (B) F5 β2m^−/−Rag1^−/− thymocytes were cultured overnight in 1 nM DEX or solvent (ethanol) and left untreated or stimulated with the indicated doses of agonist (NP68) or irrelevant (GAG) peptides presented by YO1 thymic epithelial cells. Cell lysates (4 × 10⁷ cells/lane) were immunoprecipitated with anti-CD3ε mAbs and the immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine mAbs. Membranes were then stripped and blotted with anti-CD3ζ mAbs (bottom). Similar results were obtained in three independent experiments. D, DEX.

Figure 4

DEX inhibits activation-induced TCR downregulation. Control and DEX-treated (1 μM) T cell hybridomas were stimulated by plastic-coated anti–TCR-β mAbs (H57-957) for 1 h at 37°C in 96-well culture plates. Cells were then recovered and analyzed for CD3ε expression by flow cytometry.

Figure 5

DEX does not affect the cellular distribution or the kinase activity of p56Lck and p59Fyn. (A) Cytosolic and membrane proteins from unstimulated control and DEX-treated hybridomas (4 × 10⁷ cells/condition) were separated as described in Materials and Methods. Equivalent amounts of proteins from both fractions were separated by SDS-PAGE and blotted with the indicated Abs. Lysates from DEX-treated T cell hybridomas (2 × 10⁷ cells/lane) were immunoprecipitated (IP) with (B) anti-p56Lck mAbs, (C) anti-CD4 mAbs, or (D) a rabbit serum raised against p59Fyn, and the resulting immune complexes were incubated for the indicated times at 30°C with [γ-³²P]ATP and the exogenous substrate enolase, separated by SDS-PAGE, and exposed to x-ray film for 24 h. One fourth of the immune complexes were analyzed by immunoblotting with the relevant anti-kinase Ab (bottom). The results are representative of three independent experiments. D, DEX.

Figure 6

DEX affects membrane signaling complexes. (A) GCs affect protein raft composition. Control and DEX-treated T cell hybridomas (10⁸ cells/condition) were solubilized in 1% Triton X-100 MES lysis buffer, gently sonicated, and the subsequent lysates were ultracentrifuged in a sucrose gradient as described in Materials and Methods. Undiluted GEMs and cytoplasmic fractions (1:4 dilution to avoid abnormal migration) from control and DEX-treated unstimulated cells were separated by SDS-PAGE and immunoblotted with Abs to membrane-associated (CD90, CD45, CD3ζ, p59Fyn, p56Lck, LAT) and cytoplasmic (PLCγ1) proteins. The ganglioside GM1 was detected using HRP-coupled cholera toxin B subunit. (B) Reduced phospho-LAT in the GEM fraction of DEX-treated cells. Control and DEX-treated hybridomas were left untreated or stimulated by CD3 cross-linking for 2 min and lysed in 1% Triton X-100 MES buffer. Lysates were ultracentrifuged as described previously and GEM fractions were concentrated as described in Materials and Methods, separated by SDS-PAGE, and immunoblotted (IB) with antiphosphotyrosine mAbs. Similar results were obtained in five independent experiments. (C) Coimmunoprecipitation of LAT with CD90 requires membrane cholesterol. T cell hybridomas (10⁷ cells/condition) were pretreated with 10 mM methyl-β-cyclodextrin (MCD) for 30 min at 37°C or with 0.2% saponin for 10 min at 4°C in PBS before lysis in 1% Triton X-100 MES buffer. After sonication, lysates were subjected to immunoprecipitation (IP) with anti-CD90 (clone HO-13.4)–coupled Sepharose beads. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated Abs. (D) DEX affects signaling complexes. T cell hybridomas were incubated in control and 1 μM DEX-supplemented media before lysis and immunoprecipitation with anti-CD90 mAbs. Proteins in total extracts (2 × 10⁵ cells/lane) or anti-CD90 immunoprecipitates (from 10⁷ cells/lane) were revealed by immunoblotting with the indicated Abs. D, DEX.