A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion

A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion

STAT5 Is an Ambivalent Regulator of Neutrophil Homeostasis

BACKGROUND: Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils. CONCLUSION: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

IL-6 trans-Signaling-Dependent Rapid Development of Cytotoxic CD8+ T Cell Function

Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8+ T cells that use granzyme B (GzmB) for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs) in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6) trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8+ T cells. Such LSEC-stimulated GzmB-expressing CD8+ T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8+ T cells that may be beneficial for vaccination strategies

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

Development of CD8+ T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1+ memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire

Stromal cell-specific loss of STAT5 induces prominent neutrophilia.

<p>(A) 5×10⁶ wild-type bone marrow cells were injected intravenously into lethally irradiated (1,000 rad) STAT5^−/− mice (WT→KO) and vice versa (KO→WT). 8 weeks later, serum G-CSF concentrations were measured by ELISA. Lethally irradiated wild-type mice reconstituted with wild-type bone marrow (WT→WT) and mutant mice reconstituted with mutant marrow (KO→KO) served as controls. (B) Liver sections were stained to detect G-CSF (brown). Original magnification, 250×. (C) Blood neutrophils were counted. (D) Freshly MACS-purified (T0) and 24-hr cultured (T24) blood neutrophils were assayed for apoptosis using dual color annexin-V-FITC/propidium iodide staining and flow cytometry analyses. (E–H) Mature marrow neutrophils, GMPs, CMPs, and HSCs were counted by flow cytometry. Absolute values were generated by multiplying gated percentages by total cell numbers. (I) Freshly FACS-purified marrow neutrophils, GMPs, CMPs, and HSCs were assayed for apoptosis using trypan blue exclusion.</p

STAT5^−/− mice are unable to maintain neutrophil homeostasis during cytokine-induced inflammation.

<p>(A) Wild-type (WT) and mutant mice were injected intravenously with 2 µg recombinant murine TNF-α and 2 µg recombinant murine IL-1β. Serum G-CSF concentrations were measured by ELISA before (T0) and 6 (T6), 24 (T24), and 48 (T48) hr after cytokine injection. (B) Blood neutrophils were counted at the different time points. (C) Mature marrow neutrophils (CD11c⁺Gr-1^hi cells) from control and mutant mice were counted by flow cytometry. Absolute values were generated by multiplying gated percentages by total cell numbers. (D) Blood neutrophils were isolated by MACS from wild-type and mutant mice 24 hr after cytokine injection. Freshly purified (T0) and 24-hr cultured (T24) neutrophils were assayed for apoptosis using dual color annexin-V-FITC/propidium iodide staining and flow cytometry analyses. (A–D) *, significantly different from WT values with P<0.05. °, significantly different from T0 values with P<0.05.</p

Peripheral STAT5^−/− neutrophils are functional.

<p>(A) Peritoneal neutrophils from TGA-treated wild-type (WT) or STAT5^−/− mice were analyzed for oxidation of dihydrorhodamine to fluorescent rhodamine by FACS with or without treatment with PMA (1 µg/ml). (B) STAT5^−/− neutrophils are able to produce TNF-α. 1×10⁶ peritoneal neutrophils from control or mutant mice were stimulated for 6 h with PMA (1 µg/ml), and TNF-α concentration in cell supernatants was then measured by ELISA. (C) STAT5^−/− neutrophils are phagocytic. CFSE-labeled <i>Staphylococcus aureus</i> were incubated with peritoneal neutrophils at 37°C for 30 min (T0), and gentamycin was added for an additional 30 min (T30). Cells washed free of extracellular bacteria at T0 or T30 min were analyzed by FACS for engulfed bacteria. (D) STAT5^−/− neutrophils are bactericidal. Engulfed bacteria were released by lysing peritoneal neutrophils in 1 ml water. One hundred microliters of a 1:1,000 dilution of the bacterial suspension was plated and colonies were counted after a 24-h incubation at 37°C.</p

Degradation of cytoplasmic STAT5b in WT LECs following inflammatory stimulation, and subsequent enhancement of G-CSF production.

<p>(A) Wild-type mice were injected intravenously with PBS or with 2 µg recombinant murine TNF-α and 2 µg recombinant murine IL-1β. Livers were harvested 30 min later, and liver sections were analyzed for STAT5b expression and localization by immunofluorescence and confocal microscopy as described in the Experimental Procedures. (a) STAT5b was present in the cytoplasm (green), but not the nucleus (arrows), of LECs of PBS-treated mice. (d) STAT5b was no longer detectable in LECs of cytokine-treated mice. All cell nuclei in the fields are shown by hexidium iodide staining (red, b and e), and merges of frames (a) and (b), and (d) and (e), are given in (c) and (f), respectively. Bars = 10 µm. (B) Livers collected from cytokine-treated mice were assayed for G-CSF mRNA expression by quantitative RT-PCR. Livers were harvested before cytokine injection (T0), or either 1 (T1) or 4 (T4) hr postinjection. All values are normalized to β-actin mRNA. (C) Liver sections from untreated mice (left panel) and mice treated for 4 hr with TNF-α and IL-1β (right panel) were stained to visualize G-CSF (brown). Original magnification, 250×. (D) LSECs were isolated from wild-type mice and cultured for 3 days before experiments were performed. Cells were treated for 0 (T0), 1 (T1), 3 (T3) or 6 (T6) hr with 100 IU/ml TNF-α and 500 pg/ml IL-1β. Cytoplasmic and nuclear extracts were prepared and analyzed for STAT5b expression by immunoblot. Equal loading of proteins on the gel was confirmed by probing the blots for β-actin (cytoplasmic extracts) or Oct-1 (nuclear extracts). (E) Nuclear protein extracts were assessed for STAT5 DNA-binding activity by EMSAs. Binding of Oct-1 was used as an internal standard. Nuclear extracts from prolactin-stimulated bovine MAC-T cells were used as positive controls for STAT5 activation (control). (F) G-CSF concentrations in LSEC supernatants were measured by ELISAs. *, significantly different from T0 values with P<0.05. (G) LSECs were isolated from wild-type mice and cultured for 3 days before experiments were conducted. Cells were treated with 20 µM MG132 for 30 min prior to stimulation with 100 IU/ml TNF-α and 500 pg/ml IL-1β. Cytoplasmic extracts were prepared 0 (T0), 1 (T1), 3 (T3), and 6 (T6) hr later and analyzed for STAT5b expression by immunoblot. Equal loading of proteins on the gel was confirmed by probing the blots for β-actin. (H) LSECs stimulated for 0 (T0), 1 (T1), 3 (T3), and 6 (T6) hr with 100 IU/ml TNF-α and 500 pg/ml IL-1β were assayed for STAT5b mRNA expression by real-time quantitative RT-PCR. All values were normalized to β-actin mRNA.</p