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High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids.

Strickland I, Kisich K, Hauk PJ, Vottero A, Chrousos GP, Klemm DJ, Leung DY - J. Exp. Med. (2001)

Bottom Line: GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity.Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death.Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, Colorado 80206, USA.

ABSTRACT
Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRalpha and GRbeta, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform. The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRalpha was 475 +/- 62 and 985 +/- 107 for PBMCs and neutrophils, respectively. For GRbeta, the MFI was 350 +/- 60 and 1,389 +/- 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRbeta staining to 2,497 +/- 140 (P < 0.05). No change in GRalpha expression was observed. This inversion of the GRalpha/GRbeta ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRbeta mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRalpha/GRbeta heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

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Transfection of mouse neutrophils with either pGFP or pRSh GRβ induces human GRβ protein expression. A (bright field) and B (fluorescence) show mouse neutrophils transfected with pGFP. Positive cells (green fluorescence) are observed in B. Negative cells (blue fluorescence) was visualized by counterstaining with DAPI (see Materials and Methods). Transfection efficiency was 63% in the experiment shown. Transfection efficiency was measured in two additional experiments, and found to vary from 63 to 70%. C (bright field) and D (fluorescence) show mouse neutrophils transfected with pCLeco as a control. Negative cells are observed in D (×40 objective). E are pRSh GRβ mouse neutrophils stained with a negative control antibody (rabbit IgG). Weak background staining is observed. F shows pRSh GRβ transfected neutrophils with many GRβ positively stained cells (×60 objective).
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Figure 7: Transfection of mouse neutrophils with either pGFP or pRSh GRβ induces human GRβ protein expression. A (bright field) and B (fluorescence) show mouse neutrophils transfected with pGFP. Positive cells (green fluorescence) are observed in B. Negative cells (blue fluorescence) was visualized by counterstaining with DAPI (see Materials and Methods). Transfection efficiency was 63% in the experiment shown. Transfection efficiency was measured in two additional experiments, and found to vary from 63 to 70%. C (bright field) and D (fluorescence) show mouse neutrophils transfected with pCLeco as a control. Negative cells are observed in D (×40 objective). E are pRSh GRβ mouse neutrophils stained with a negative control antibody (rabbit IgG). Weak background staining is observed. F shows pRSh GRβ transfected neutrophils with many GRβ positively stained cells (×60 objective).

Mentions: Transient expression of human GRβ in primary murine neutrophils required development of a method to efficiently introduce plasmid DNA into the cells. To accomplish this, we relied on transient permeabilization of the cells with streptolysin O, which has been employed in a similar manner by others 2223. Permeabilization of the neutrophils in the presence of plasmid DNA encoding GFP under control of the CMV immediate early promoter resulted in efficient uptake and expression of the plasmid (Fig. 7). Plasmid transfection efficiency was 63 ± 10% (mean ± SEM) as assessed by measuring the percentage of cells expressing GFP (Fig. 7 B). Cells that had been treated with streptolysin O in the presence of a control plasmid, which did not encode GFP, showed no fluorescence (Fig. 7 D).


High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids.

Strickland I, Kisich K, Hauk PJ, Vottero A, Chrousos GP, Klemm DJ, Leung DY - J. Exp. Med. (2001)

Transfection of mouse neutrophils with either pGFP or pRSh GRβ induces human GRβ protein expression. A (bright field) and B (fluorescence) show mouse neutrophils transfected with pGFP. Positive cells (green fluorescence) are observed in B. Negative cells (blue fluorescence) was visualized by counterstaining with DAPI (see Materials and Methods). Transfection efficiency was 63% in the experiment shown. Transfection efficiency was measured in two additional experiments, and found to vary from 63 to 70%. C (bright field) and D (fluorescence) show mouse neutrophils transfected with pCLeco as a control. Negative cells are observed in D (×40 objective). E are pRSh GRβ mouse neutrophils stained with a negative control antibody (rabbit IgG). Weak background staining is observed. F shows pRSh GRβ transfected neutrophils with many GRβ positively stained cells (×60 objective).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193396&req=5

Figure 7: Transfection of mouse neutrophils with either pGFP or pRSh GRβ induces human GRβ protein expression. A (bright field) and B (fluorescence) show mouse neutrophils transfected with pGFP. Positive cells (green fluorescence) are observed in B. Negative cells (blue fluorescence) was visualized by counterstaining with DAPI (see Materials and Methods). Transfection efficiency was 63% in the experiment shown. Transfection efficiency was measured in two additional experiments, and found to vary from 63 to 70%. C (bright field) and D (fluorescence) show mouse neutrophils transfected with pCLeco as a control. Negative cells are observed in D (×40 objective). E are pRSh GRβ mouse neutrophils stained with a negative control antibody (rabbit IgG). Weak background staining is observed. F shows pRSh GRβ transfected neutrophils with many GRβ positively stained cells (×60 objective).
Mentions: Transient expression of human GRβ in primary murine neutrophils required development of a method to efficiently introduce plasmid DNA into the cells. To accomplish this, we relied on transient permeabilization of the cells with streptolysin O, which has been employed in a similar manner by others 2223. Permeabilization of the neutrophils in the presence of plasmid DNA encoding GFP under control of the CMV immediate early promoter resulted in efficient uptake and expression of the plasmid (Fig. 7). Plasmid transfection efficiency was 63 ± 10% (mean ± SEM) as assessed by measuring the percentage of cells expressing GFP (Fig. 7 B). Cells that had been treated with streptolysin O in the presence of a control plasmid, which did not encode GFP, showed no fluorescence (Fig. 7 D).

Bottom Line: GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity.Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death.Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, Colorado 80206, USA.

ABSTRACT
Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRalpha and GRbeta, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform. The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRalpha was 475 +/- 62 and 985 +/- 107 for PBMCs and neutrophils, respectively. For GRbeta, the MFI was 350 +/- 60 and 1,389 +/- 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRbeta staining to 2,497 +/- 140 (P < 0.05). No change in GRalpha expression was observed. This inversion of the GRalpha/GRbeta ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRbeta mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRalpha/GRbeta heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

Show MeSH
Related in: MedlinePlus