1. Synthesis of glucocorticoid-induced leucine zipper (GILZ) by macrophages: an anti-inflammatory and immunosuppressive mechanism shared by glucocorticoids and IL-10
by Dominique Berrebi, Stefano Bruscoli, Nicolas Cohen, Arnaud Foussat, Graziella Migliorati, Laurence Bouchet-Delbos, Marie-Christine Maillot, Alain Portier, Jacques Couderc, Pierre Galanaud, Michel Peuchmaur, Carlo Riccardi, and Dominique Emilie
Blood
Volume 101(2):
January 15, 2003
©2003 by American Society of Hematology

2. GILZ gene expression in normal human tissues
GILZ gene expression in normal human tissues.GILZ gene expression was studied by in situ hybridization in the lungs (A-B), liver (C), and kidneys (D), with either an antisense (A, C-D) or a sense (B) GILZ probe.
GILZ gene expression in normal human tissues.GILZ gene expression was studied by in situ hybridization in the lungs (A-B), liver (C), and kidneys (D), with either an antisense (A, C-D) or a sense (B) GILZ probe. In lung samples, macrophages were identified by immunohistochemistry with anti-CD68 antibodies (E), and GILZ gene–expressing cells were identified by double-labeling experiments in which in situ hybridization with use of the GILZ antisense probe (dark blue) was combined with immunohistochemistry with an anti-CD68 antibody (brown; F). The results shown are representative of experiments performed on 3 different lungs, livers, and kidneys. Original magnifications A-B, × 400; C, × 250; inset C, × 400; D, × 400; E-F, × 1600; inset F, × 2000.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

3. In vitro induction of GILZ by GCs
In vitro induction of GILZ by GCs.Production of GILZ by human monocytes cultured alone or with DXM was studied by RT-PCR (A-B) and by Western blotting (C).
In vitro induction of GILZ by GCs.Production of GILZ by human monocytes cultured alone or with DXM was studied by RT-PCR (A-B) and by Western blotting (C). (A) GILZ RT-PCR, 3 experiments. (B) Optical density of products amplified from GILZ mRNA (mean ± SEM) in these 3 experiments. (C) GILZ protein in cells cultured without (lane 1) or with (lane 2) DXM. Results from 1 experiment representative of 2 are shown.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Kinetics of GILZ gene induction
Kinetics of GILZ gene induction.(A) The kinetics of GILZ gene induction was determined in THP-1 cells stimulated with DXM. Results are expressed as means ± SEMs of 3 experiments.
Kinetics of GILZ gene induction.(A) The kinetics of GILZ gene induction was determined in THP-1 cells stimulated with DXM. Results are expressed as means ± SEMs of 3 experiments. (B) Requirement for protein synthesis was determined using THP-1 cells cultured with or without CHX and stimulated for 4 hours with DXM. Results are expressed as means ± SEMs of 2 experiments.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

5. IL-10 and Th2 cytokines stimulate GILZ production by human monocytes
IL-10 and Th2 cytokines stimulate GILZ production by human monocytes.(A) GILZ mRNA from monocytes cultured with or without IL-10, IL-4, or IL-13 was amplified by RT-PCR.
IL-10 and Th2 cytokines stimulate GILZ production by human monocytes.(A) GILZ mRNA from monocytes cultured with or without IL-10, IL-4, or IL-13 was amplified by RT-PCR. Optical density of amplified product was determined, and each is expressed as the mean ± SEM of 3 experiments. (B) GILZ protein in Western blot studies with monocytes cultured alone or with IL-4, IL-10, or IL-13. Results from 1 experiment typical of 2.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

6. In vivo induction of GILZ in mice
In vivo induction of GILZ in mice.(A) Production of GILZ was evaluated in mice treated with PBS (control, n = 6) or with DXM (n = 6).
In vivo induction of GILZ in mice.(A) Production of GILZ was evaluated in mice treated with PBS (control, n = 6) or with DXM (n = 6). GILZ gene expression was measured by RT-PCR. (i) Optical density of products amplified from GILZ mRNA (means ± SEMs), *P < .01 (Mann-WhitneyU test). ■ indicates control; ▪, treated with DXM. (ii) A typical result for analysis of GILZ gene expression in the lung from 3 control and 3 DXM-treated mice. 0: negative control, with no cDNA. (iii) Presence of GILZ and β-tubulin in the lung of control and DXM-treated mice, as analyzed by Western blotting. Results shown are from 1 experiment representative of 2. (B) In vitro and (C) in vivo GILZ gene expression by peritoneal macrophages were evaluated by RNase protection assay (RPA). (Bi, Ci) Lanes 1 and 2: undigested β-actin and GILZ probes, respectively; lane 3: RPA in the presence of yeast RNA. (Bi) Lanes 4 and 5: macrophages cultured alone and with DXM, respectively. (Ci) Lanes 4 and 5: macrophages from control and DXM-treated mice, respectively. The ratios between protected GILZ and β-actin probes for lanes 4 and 5 are shown in panels Bii and Cii.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

7. GILZ gene expression in IL-10 transgenic mice
GILZ gene expression in IL-10 transgenic mice.Expression of Mac-1 (A,C) and GILZ gene (B,D) was analyzed by immunohistochemistry and by in situ hybridization, respectively, in BALB/C mice (A-B) and in IL-10 transgenic mice (C-D).
GILZ gene expression in IL-10 transgenic mice.Expression of Mac-1 (A,C) and GILZ gene (B,D) was analyzed by immunohistochemistry and by in situ hybridization, respectively, in BALB/C mice (A-B) and in IL-10 transgenic mice (C-D). Original magnifications A, × 100; B, × 160; C-D, × 250.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

8. GILZ inhibits CD80 and CD86 expression
GILZ inhibits CD80 and CD86 expression.(A) THP-1 cells were cultured with and without DXM or IL-10.
GILZ inhibits CD80 and CD86 expression.(A) THP-1 cells were cultured with and without DXM or IL-10. (B) THP-1 cells were transfected with the control (pcDNA3) vector or with the GILZ-encoding vector. CD80 (left panels) and CD86 (right panels) expression was determined by flow cytometry at the end of culture. The dashed curves correspond to the staining obtained with the control mAb. Labeling with anti-CD80 or anti-CD86 antibody is shown by the open histograms and solid histograms for cells cultured without and with IFNγ, respectively. Results shown are from 1 experiment of 4. In the upper right-hand corner of each histogram is the mean fraction (%) of cells in which IFNγ stimulated CD80 or CD86 expression in these 4 experiments.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

9. GILZ inhibits chemokine production
GILZ inhibits chemokine production.THP-1 cells were transfected with either a control plasmid or GILZ-pcDNA3 (GILZ).
GILZ inhibits chemokine production.THP-1 cells were transfected with either a control plasmid or GILZ-pcDNA3 (GILZ). (A) RANTES and (B) MIP-1α concentrations in the supernatants. (C) RT-PCR experiments for GILZ, RANTES, and β-actin. The results are presented as means ± SEMs of 3 experiments, with 100% corresponding to unstimulated control cells. The concentrations (means ± SEMs) of RANTES and MIP-1α in these control cells were 1130 ± 768 pg/mL and 5172 ± 1452 pg/mL, respectively.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

10. GILZ inhibits TLR2 expression
GILZ inhibits TLR2 expression.(A) THP-1 cells were cultured with or without DXM or IL-10.
GILZ inhibits TLR2 expression.(A) THP-1 cells were cultured with or without DXM or IL-10. (B) THP-1 cells were transfected with the control (pcDNA3) or the GILZ-encoding vector. Cells were stimulated with LPS, IFNγ, or an anti-CD40 mAb. TLR2 expression was determined at the end of culture. Open histograms correspond to the staining obtained for the control Ab, and solid histograms correspond to cells labeled with the anti-TLR2 Ab. Results are from 1 experiment of 4. In the upper right-hand corner of each histogram, the mean fraction (%) of cells expressing TLR2 in these 4 experiments is shown. In cells cultured without LPS, IFNγ, or anti-CD40 mAb, 6.3% ± 1.0% of the cells expressed TLR2.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

11. DXM, IL-10, or Th2 cytokine treatment of THP-1 cells induces the association of GILZ with p65.(A-B) THP-1 cells were cultured for 4 hours alone or with DXM, IL-4, IL-10, or IL-13.
DXM, IL-10, or Th2 cytokine treatment of THP-1 cells induces the association of GILZ with p65.(A-B) THP-1 cells were cultured for 4 hours alone or with DXM, IL-4, IL-10, or IL-13. Cellular protein was immunoprecipitated with anti-p65 antiserum. Immunoblotting was performed with anti-p65 (A) or anti-GILZ (B) antibody. HC: heavy chain of the antibody used in the assay. (C) THP-1 cells were transfected either with the pcDNA3 control vector alone or with the NFkB-luciferase vector, with or without the GILZ gene–encoding vector. Luciferase activity was determined 4 hours after stimulation with LPS. Results are from 1 experiment typical of 3. Luciferase activity for cells transfected with pcDNA3 alone was < 10 units.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology

12. GILZ gene expression in DTH reactions and in Burkitt lymphomas
GILZ gene expression in DTH reactions and in Burkitt lymphomas.RANTES (A) and GILZ (B,E) gene expression was analyzed by in situ hybridization with an antisense probe in patients with DTH reactions (A-B) and in Burkitt lymphomas (E).
GILZ gene expression in DTH reactions and in Burkitt lymphomas.RANTES (A) and GILZ (B,E) gene expression was analyzed by in situ hybridization with an antisense probe in patients with DTH reactions (A-B) and in Burkitt lymphomas (E). No labeling was observed with the sense probe (shown in panel F for Burkitt lymphoma). (C-D) Hematoxylin-eosin staining and CD68 immunostaining of a Burkitt lymphoma, respectively. The results shown in panels A and B correspond to a patient with Crohn disease; G indicates granuloma; EG, extragranuloma area. In Panels C and E, arrows indicate tumor-infiltrating macrophages containing apoptotic bodies. Original magnifications A, × 400; inset A, × 250; B, × 160; C, × 1000; D-E × 1600; F, × 1000.
Dominique Berrebi et al. Blood 2003;101:
©2003 by American Society of Hematology