Yun Cao

Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms.

Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids.

BCL-2 protects human and mouse Th17 cells from glucocorticoid-induced apoptosis.

Glucocorticoid resistance has been associated with Th17‐driven inflammation, the mechanisms of which are not clear. We determined whether human and mouse Th17 cells are resistant to glucocorticoid‐induced apoptosis.

Determinants of the heightened activity of glucocorticoid receptor translational isoforms.

Translational isoforms of the glucocorticoid receptor α (GR-A, -B, -C1, -C2, -C3, -D1, -D2, and -D3) have distinct tissue distribution patterns and unique gene targets. The GR-C3 isoform-expressing cells are more sensitive to glucocorticoid killing than cells expressing other GRα isoforms and the GR-D isoform-expressing cells are resistant to glucocorticoid killing. Whereas a lack of activation function 1 (AF1) may underlie the reduced activity of the GR-D isoforms, it is not clear how the GR-C3 isoform has heightened activity. Mutation analyses and N-terminal tagging demonstrated that steric hindrance is probably the mechanism for the GR-A, -B, -C1, and -C2 isoforms to have lower activity than the GR-C3 isoform. In addition, truncation scanning analyses revealed that residues 98 to 115 are critical in the hyperactivity of the human GR-C3 isoform. Chimera constructs linking this critical fragment with the GAL4 DNA-binding domain showed that GR residues 98 to 115 do not contain any independent transactivation activity. Mutations at residues Asp101 or Gln106 and Gln107 all reduced the activity of the GR-C3 isoform. In addition, functional studies indicated that Asp101 is crucial for the GR-C3 isoform to recruit coregulators and to mediate glucocorticoid-induced apoptosis. Thus, charged and polar residues are essential components of an N-terminal motif that enhances the activity of AF1 and the GR-C3 isoform. These studies, together with the observations that GR isoforms have cell-specific expression patterns, provide a molecular basis for the tissue-specific functions of GR translational isoforms.

Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells

Background Sialic acid–binding immunoglobulin‐like lectin (Siglec)‐8 is a cell‐surface protein expressed selectively on human eosinophils, mast cells, and basophils, making it an ideal target for the treatment of diseases involving these cell types. However, the effective delivery of therapeutic agents to these cells requires an understanding of the dynamics of Siglec‐8 surface expression. Objectives We sought to determine whether Siglec‐8 is endocytosed in human eosinophils and malignant mast cells, identify mechanisms underlying its endocytosis, and demonstrate whether a toxin can be targeted to Siglec‐8–bearing cells to kill these cells. Methods Siglec‐8 surface dynamics were examined by flow cytometry using peripheral blood eosinophils, mast cell lines, and Siglec‐8–transduced cells in the presence of inhibitors targeting components of endocytic pathways. Siglec‐8 intracellular trafficking was followed by confocal microscopy. The ribosome‐inhibiting protein saporin was conjugated to a Siglec‐8–specific antibody to examine the targeting of an agent to these cells through Siglec‐8 endocytosis. Results Siglec‐8 endocytosis required actin rearrangement, tyrosine kinase and protein kinase C activities, and both clathrin and lipid rafts. Internalized Siglec‐8 localized to the lysosomal compartment. Maximal endocytosis in Siglec‐8–transduced HEK293T cells required an intact immunoreceptor tyrosine‐based inhibitory motif. Siglec‐8 was also shuttled to the surface via a distinct pathway. Sialidase treatment of eosinophils revealed that Siglec‐8 is partially masked by sialylated cis ligands. Targeting saporin to Siglec‐8 consistently caused extensive cell death in eosinophils and the human mast cell leukemia cell line HMC‐1.2. Conclusions Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec‐8 endocytic pathway.

Sialic acid–binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating β2-integrin–dependent function in human eosinophils

Background: Siglec‐8 is a CD33 subfamily cell‐surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec‐8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33‐related Siglecs function as inhibitory receptors, but the ability of Siglec‐8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec‐8 might instead function as an activating receptor. Objective: We sought to determine the role of IL‐5 priming and identify the signaling molecules involved in Siglec‐8 function for human eosinophils. Methods: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec‐8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec‐8 function for human eosinophils. Results: Cytokine priming was required to elicit the unanticipated finding that Siglec‐8 engagement promotes rapid &bgr;2‐integrin–dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec‐8–mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec‐8–mediated eosinophil apoptosis. Finally, engagement of Siglec‐8 on IL‐5–primed eosinophils resulted in increased phosphorylation of Akt, p38, and c‐Jun N‐terminal kinase 1 that was also &bgr;2‐integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec‐8–mediated eosinophil apoptosis. Conclusions: These data demonstrate that Siglec‐8 functions uniquely as an activating receptor on IL‐5–primed eosinophils through a novel pathway involving regulation of &bgr;2‐integrin–dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Immunopathology alters Th17 cell glucocorticoid sensitivity

Th17 cells contribute to several inflammatory conditions and increasing evidence supports that Th17 cells are glucocorticoid resistant. However, Th17 cells in psoriasis and related diseases are glucocorticoid sensitive. We compare glucocorticoid sensitive and resistant immunological diseases and suggest that several aspects in Th17‐related diseases alter glucocorticoid sensitivity of Th17 cells. We identify molecular pathways that are implicated in glucocorticoid sensitivity of Th17 cells in the literature, as this information is useful for developing approaches to overcome glucocorticoid‐resistant immunopathology.

Frontline Science: Characterization of a novel mouse strain expressing human Siglec‐8 only on eosinophils

Sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐8 is a human cell surface protein expressed exclusively on eosinophils, mast cells, and basophils that, when engaged, induces eosinophil apoptosis and inhibits mast cell mediator release. This makes Siglec‐8 a promising therapeutic target to treat diseases involving these cell types. However, preclinical studies of Siglec‐8 targeting in vivo are lacking because this protein is only found in humans, apes, and some monkeys. Therefore, we have developed a mouse strain in which SIGLEC8 transcription is activated by Cre recombinase and have crossed this mouse with the eoCre mouse to achieve eosinophil‐specific expression. We confirmed that Siglec‐8 is expressed exclusively on the surface of mature eosinophils in multiple tissues at levels comparable to those on human blood eosinophils. Following ovalbumin sensitization and airway challenge, Siglec‐8 knock‐in mice generated a pattern of allergic lung inflammation indistinguishable from that of littermate controls, suggesting that Siglec‐8 expression within the eosinophil compartment does not alter allergic eosinophilic inflammation. Using bone marrow from these mice, we demonstrated that, during maturation, Siglec‐8 expression occurs well before the late eosinophil developmental marker C‐C motif chemokine receptor 3, consistent with eoCre expression. Antibody ligation of the receptor induces Siglec‐8 endocytosis and alters the phosphotyrosine profile of these cells, indicative of productive signaling. Finally, we demonstrated that mouse eosinophils expressing Siglec‐8 undergo cell death when the receptor is engaged, further evidence that Siglec‐8 is functional on these cells. These mice should prove useful to investigate Siglec‐8 biology and targeting in vivo in a variety of eosinophilic disease models.

Granulocyte colony-stimulating factor blockade enables dexamethasone to inhibit lipopolysaccharide-induced murine lung neutrophils

Glucocorticoids promote neutrophilic inflammation, the mechanisms of which are poorly characterized. Using a lipopolysaccharide (LPS)-induced acute murine lung injury model, we determined the role of granulocyte colony-stimulating factor (G-CSF) in mouse lung neutrophil numbers in the absence and presence of dexamethasone, a potent glucocorticoid. G-CSF was blocked using a neutralizing antibody. Airway neutrophil numbers, cytokine levels, and lung injury parameters were measured. Glucocorticoid treatment maintained LPS-induced airway G-CSF while suppressing TNF and IL-6. The addition of anti-G-CSF antibodies enabled dexamethasone to decrease airway G-CSF, neutrophils, and lung injury scores. In LPS-challenged murine lungs, structural cells and infiltrating leukocytes produced G-CSF. In vitro using BEAS 2B bronchial epithelial cells, A549 lung epithelial cells, human monocyte-derived macrophages, and human neutrophils, we found that dexamethasone and proinflammatory cytokines synergistically induced G-CSF. Blocking G-CSF production in BEAS 2B cells using shRNAs diminished the ability of BEAS 2B cells to protect neutrophils from undergoing spontaneous apoptosis. These data support that G-CSF plays a role in upregulation of airway neutrophil numbers by dexamethasone in the LPS-induced acute lung injury model.