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

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

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

Increased granulopoiesis in STAT5^−/− mice.

<p>(A) Apoptosis of peripheral neutrophils. MACS-purified blood peripheral neutrophils from wild-type (WT) and STAT5^−/− mice were cultured for 24 hr in the absence or presence of 0.5 or 25 ng/ml G-CSF or 25 ng/ml GM-CSF. 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. *, significantly different from T0 values with P<0.05. °, significantly different from WT values with P<0.05. (B) Bone marrow was harvested from both hind limbs of either WT or STAT5^−/− mice, and cell counts were determined after red blood cell lysis. (C–F) Mature marrow neutrophils (CD11c⁺Gr-1^hi cells), GMPs (Lin⁻Sca-1⁻IL-7Rα⁻c-kit⁺CD34⁺FcγR⁺ cells), CMPs (Sca-1⁻IL-7Rα⁻c-kit⁺CD34⁺FcγR^low cells), and HSCs (Lin⁻Sca-1^hiIL-7Rα⁻c-kit^hi) from the bone marrow of control and mutant mice were counted by flow cytometry. Absolute values were generated by multiplying gated percentages by total cell numbers. (G) Freshly FACS-purified marrow neutrophils, GMPs, CMPs, and HSCs were assayed for apoptosis using trypan blue exclusion. (B–G) *, significantly different from WT values with P<0.05.</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

Characterization of wild-type and STAT5^−/− C57Bl/6 mice.

<p>(A) Tail DNA from STAT5^+/+, STAT5^+/−, and STAT5^−/− C57Bl/6 mice was prepared and PCR were performed to amplify wild-type <i>stat5a</i> and <i>stat5b</i> DNA as well as the TK cassette. The primers are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000727#s4" target="_blank">Material and methods</a> section. (B) LSECs were isolated from wild-type (WT) and STAT5^−/− mice. Whole-cell extracts were prepared and analyzed for STAT5b and STAT5bΔN expression by immunoblot. We used an antibody (G-2) directed to an epitope mapping at the C-terminus of STAT5b. Equal loading of proteins on the gel was confirmed by probing the blots for β-actin. (C) Blood neutrophils were isolated from WT and STAT5^−/− mice. Whole-cell extracts were prepared and analyzed for STAT5b/STAT5bΔN (left panel) and STAT5a/STAT5aΔN (right panel) expression by immunoblot. For STAT5b/STAT5bΔN, we used the G-2 antibody. For STAT5a/STAT5aΔN, we used an antibody (L-20) raised against a peptide mapping at the C-terminus of STAT5a. Equal loading of proteins on the gel was confirmed by probing the blots for β-actin.</p