By comparing mRNA species expressed in dexamethasone (DEX)-treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.

GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8 + and CD4 + peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2R § ) and production of IL-2 and IFN-+ were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4 + CD25 + regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR -/-CD4 + CD25 + and GITR +/+ CD4 + CD25 + cells were equally able to exert suppressor activity on CD4 + CD25 -responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

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Glucocorticoids and interleukin 10 (IL-10) prevent macrophage activation. In murine lymphocytes, glucocorticoids induce expression of glucocorticoid-induced leucine zipper (GILZ), which prevents the nuclear factor B (NF-B)-mediated activation of transcription. We investigated whether GILZ could account for the deactivation of macrophages by glucocorticoids and IL-10. We found that GILZ was constitutively produced by macrophages in nonlymphoid tissues of humans and mice. Glucocorticoids and IL-10 stimulated the production of GILZ by macrophages both in vitro and in vivo. Transfection of the macrophagelike cell line THP-1 with the GILZ gene inhibited the expression of CD80 and CD86 and the production of the proinflammatory chemokines regulated on activation normal T-cell expressed and secreted (CCL5) and macrophage inflammatory protein 1␣ (CCL3). It also prevented toll-like receptor 2 production induced by lipopolysaccharide, interferon␥, or an anti-CD40 mAb, as well as NF-B function. In THP-1 cells treated with glucocorticoids or IL-10, GILZ was associated with the p65 subunit of NF-B. Activated macrophages in the granulomas of patients with Crohn disease or tuberculosis do not produce GILZ. In contrast, GILZ production persists in tumorinfiltrating macrophages in Burkitt lymphomas. Therefore, GILZ appears to play a key role in the anti-inflammatory and immunosuppressive effects of glucocorticoids and IL-10. Glucocorticoid treatment stimulates GILZ production, reproducing an effect of IL-10, a natural anti-inflammatory agent. The development of delayedtype hypersensitivity reactions is associated with the down-regulation of GILZ gene expression within lesions. In contrast, the persistence of GILZ gene expression in macrophages infiltrating Burkitt lymphomas may contribute to the failure of the immune system to reject the tumor. IntroductionGlucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive drugs. Their therapeutic effects are largely due to their ability to inhibit many functions of macrophages and of other antigen-presenting cells. Interleukin 10 (IL-10) is an antiinflammatory cytokine that has a number of effects in common with GCs, particularly those affecting macrophage functions. Both GCs 1-13 and IL-10 (reviewed in Stordeur and Goldman 14 ) inhibit antigen processing, the expression of HLA, CD80, and CD86, and the synthesis of nitric oxide, cyclo-oxygenase 2, adhesion molecules, cytokines, and chemokines. The intracellular events induced by the binding of GCs and IL-10 to their respective receptors are not fully understood, but they also share certain characteristics. In particular, both GCs and IL-10 interfere with the function of the transcriptional activators AP-1 and NF-B (reviewed in Stordeur and Goldman, 14 Karin, 15 and Barnes and Karin 16 ). Pathogenassociated molecular patterns (PAMPs) of bacterial components activate macrophages by binding to the toll-like receptors (TLRs), which trigger the nuclear factor B (NF-B) pathway and stimulate the production of inflammatory protein...

Glucocorticoid hormones regulate essential body functions in mammals, control cell metabolism, growth, differentiation, and apoptosis. Importantly, they are potent suppressors of inflammation, and multiple immune-modulatory mechanisms involving leukocyte apoptosis, differentiation, and cytokine production have been described. Due to their potent anti-inflammatory and immune-suppressive activity, synthetic glucocorticoids (GCs) are the most prescribed drugs used for treatment of autoimmune and inflammatory diseases. It is long been noted that males and females exhibit differences in the prevalence in several autoimmune diseases (AD). This can be due to the role of sexual hormones in regulation of the immune responses, acting through their endogenous nuclear receptors to mediate gene expression and generate unique gender-specific cellular environments. Given the fact that GCs are the primary physiological anti-inflammatory hormones, and that sex hormones may also exert immune-modulatory functions, the link between GCs and sex hormones may exist. Understanding the nature of this possible crosstalk is important to unravel the reason of sexual disparity in AD and to carefully prescribe these drugs for the treatment of inflammatory diseases. In this review, we discuss similarities and differences between the effects of sex hormones and GCs on the immune system, to highlight possible axes of functional interaction.

Glucocorticoid-induced leucine zipper (GILZ) is a 137 amino acid protein, rapidly induced by treatment with glucocorticoids (GC), characterized by a leucine zipper (LZ) domain (76–97 amino acids), an N-terminal domain (1–75 amino acids) and a C-terminal PER domain (98–137 amino acids) rich in proline and glutamic acid residues. We have previously shown that GILZ binds to and inhibits NF-κB activity. In the present study we used a number of mutants with the aim of defining the GILZ molecular domains responsible for GILZ/p65NF-κB interaction. Results, obtained by in vitro and in vivo co-immunoprecipitation (Co-IP) and by transcriptional activity experiments, indicate that GILZ homo-dimerization, through the LZ domain, as well as the C-terminal PER domain, particularly the 121–123 amino acids, are both necessary for GILZ interaction with NF-κB, inhibition of transcriptional activity and of IL-2 synthesis.

Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)–GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ−/− mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression.

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) control immune responses and prevent autoimmunity. Treatment with glucocorticoids (GCs) has been shown to increase Treg cell frequency, but the mechanisms of their action on Treg cell induction are largely unknown. Here, we report that glucocorticoid-induced leucine zipper (GILZ), a protein induced by GCs, promotes Treg cell production. In mice, GILZ overexpression causes an increase in Treg cell number, whereas GILZ deficiency results in impaired generation of peripheral Treg cells (pTreg), associated with increased spontaneous and experimental intestinal inflammation. Mechanistically, we found that GILZ is required for GCs to cooperate with TGF-β in FoxP3 induction, while it enhances TGF-β signaling by binding to and promoting Smad2 phosphorylation and activation of FoxP3 expression. Thus, our results establish an essential GILZ-mediated link between the anti-inflammatory action of GCs and the regulation of TGF-β-dependent pTreg production.