Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Treating rat primary microglia for 6 hr with 20 ng/ml IL4 or 20 ng/ml IL10 resulted in gene expression changes that were specific for IL4 ( A ), specific for IL10 ( B ), or common to both treatments ( C ). mRNA expression was normalized to the housekeeping gene, HPRT1 , and shown as mean ± SEM with the number of individual cultures indicated on each bar. [Note the differing Y-axis scales.] One-way ANOVA with Tukey's post-hoc test revealed differences from unstimulated (control) microglia: * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Gene Expression, Expressing, Control

Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: A. Representative fluorescence micrographs show podonut expression in unstimulated primary rat microglia or 24 hr after treatment with LPS (10 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). The arrows indicate examples of a single, large donut-shaped ring of podosomes, which we call a ‘podonut’ in the lamellum of unipolar microglia. Cells were stained for filamentous (F-) actin with phalloidin (green) to quantify the proportion of cells with a podonut, and with the nuclear marker, DAPI (blue) to quantify total cell numbers. Scale bar, 50 µm. B. Podosome (podonut) expression is affected by the microglial activation state. The proportion of microglial cells bearing a podonut in the lamellum (sum of 3 randomly selected fields of view at 10× magnification) was normalized to unstimulated cells. C–E. Microglia were unstimulated or stimulated for 24 hr with 20 ng/ml IL4 or IL10, and then exposed for 6 hr to control medium or the KCa2.3 inhibitors, 5 nM tamapin or 7 µM NS8593, which also inhibits TRPM7 channels (see Results and , ). All graphical data are shown as mean ± SEM with sample size (# of individual cultures) indicated on each bar. A one-way ANOVA with Tukey's post-hoc analysis was used to determine significant differences. * p <0.05; *** p <0.001.

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Fluorescence, Expressing, Staining, Marker, Activation Assay, Control

Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: A. Microglia in the upper well of Transwells were unstimulated or stimulated with either 20 ng/ml IL4 or 20 ng/ml IL10 for 24 hr. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells, indicated by the dashed line. B–D. Microglia were unstimulated or stimulated with IL4 or IL10 as in panel A, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, 7 µM NS8593 or 10 µM AA-861. E–H. IL4 and IL10 increase the invasion capacity of rat microglia, and TRPM7 is involved. Microglia were plated in the upper wells of Matrigel chambers, with or without stimulation by 20 ng/ml IL4 or 20 ng/ml IL10 for 24 hr. E. Invasion of control (unstimulated) microglia was compared with IL4- and IL10-treated cells. F–H. Microglia were unstimulated or stimulated with IL4 or IL10 as in panel A, with or without simultaneous addition of a channel inhibitor: 100 nM apamin, 7 µM NS8593 or 10 µM AA-861. For each stimulus, the number of cells that had migrated or invaded was normalized to the level without a channel inhibitor. The dashed line in all graphs indicates the level in control (unstimulated) cells. Data are expressed as mean ± SEM with the number of individual cultures indicated on each bar. A one-way ANOVA with Tukey's post-hoc test was used to compare results with and without a channel inhibitor; * p <0.05, ** p <0.01, *** p <0.001; or for NS8593 versus AA-861 (†p<0.05).

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: A. Representative fluorescence micrographs show MLS-9 cells (rat microglial cell line), unstimulated or 24 hr after stimulation with LPS (100 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). Cells were stained for F-actin with phalloidin (green) and the nuclear marker, DAPI (blue). Most cells were bipolar with membrane ruffling at one end (examples shown by arrows). Scale bar, 50 µm. B. MLS-9 microglial cells in the upper well of Transwells were unstimulated, or stimulated (24 hr) with 20 or 100 ng/ml IL4, or with 20 or 100 ng/ml IL10. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells. The dashed line in both graphs indicates the level in control (unstimulated) cells. C. Cells were stimulated as in panel B, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, or 7 µM NS8593. For each stimulus, the number of cells that had migrated was normalized to the level without a channel inhibitor. Data are expressed as mean ± SEM with the number of individual cultures indicated on each bar. In panel B, a two-way ANOVA with Bonferroni post-hoc analysis was used to determine significant differences between stimulation (*) and dose (†). In panel C, A one-way ANOVA with Tukey's post-hoc test was used to determine treatment effects. One symbol, p <0.05; two symbols, p <0.01; three symbols, p <0.001.

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Fluorescence, Staining, Marker, Membrane, Control

Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: A. Expression of KCNN3 mRNA was quantified using NanoString nCounter analysis in unstimulated primary microglia, and at 6 hr (solid bars) and 24 hr (striped bars) after treatment with 20 ng/ml IL4- or IL10-treatment. B–D. NS8593 inhibits the KCa2.3 current, while AA-861 has no effect. KCa2.3 currents were recorded from MLS-9 microglial cells in the perforated-patch configuration produced by amphotericin B (200 µg/ml) in the pipette. The voltage protocol throughout was a 120 ms-long voltage ramp from −100 to +80 mV from a holding potential of −70 mV. The bath always contained 1 µM TRAM-34 to block KCa3.1 currents. Riluzole (300 µM) was used simply as a tool to activate the KCa2.3 current . B. Upper panel : Representative traces show the current evoked by riluzole with or without 7 µM NS8593. Lower panel : The representative time course (current measured at +80 mV) illustrates KCa2.3 current activation and its inhibition by NS8593. C. Representative currents and time course show that no current was activated when 7 µM NS8593 was present in the bath. D. The current is insensitive to 10 µM AA-861. Note that current activation by riluzole is readily reversible (wash), as we previously showed . E–G. The KCa2.3 current in primary rat microglial cells is inhibited by 7 µM NS8593 under differing activation states. Currents were recorded using the same patch-clamp configuration as described for MLS-9 cells. Upper panels : Representative currents in microglia that were unstimulated (E), or treated for 24 hr with 20 ng/mL IL4 (F) or 20 ng/mL IL10 (G). Lower panels : A representative time course for each cell (current at +80 mV) shows activation by riluzole and inhibition by NS8593.

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Produced, Transferring, Blocking Assay, Activation Assay, Inhibition, Patch Clamp

Journal: PLoS ONE

Article Title: Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: A. TRPM7 mRNA expression was measured using quantitative real-time RT-PCR at 6 hr (solid bars) and 24 hr (striped bars) in unstimulated rat microglia or after treatment with 20 ng/ml IL4 or 20 ng/ml IL10. Values are expressed as mean expression (normalized to HPRT1) ± SEM, with the number of individual cultures indicated on each bar. * p <0.05 indicates the difference from time-matched control (unstimulated) cells, and was determined using a 2-way ANOVA followed by Bonferroni post-hoc test. B–E. The TRPM7 current and block by NS8593 were not affected by 24 hr treatment with 20 ng/ml IL4 or IL10. Whole-cell recordings were performed on primary rat microglia using Mg 2+ -free pipette solution. B, C. Representative currents (upper panels) in response to 120 ms-long ramps from −100 to +115 mV, from a holding potential of −10 mV. Time course of the current (lower panels) measured at +115 mV. D, E. Summary of TRPM7 current amplitudes measured at +115 mV ( D ) and percent inhibition of TRPM7 currents by 7 µM NS8593 ( E ). Graphical data are presented as mean ± SEM for the number of cells indicated on each bar, and were compared using a one-way ANOVA with Tukey's post-hoc test.

Article Snippet: Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Blocking Assay, Transferring, Inhibition

Journal:

Article Title: Cellular and Cytokine Correlates of Mucosal Protection in Murine Model of Oral Candidiasis

doi:

Figure Lengend Snippet: IL-4 and IFN-γ mRNA gene expression in CLN cells. Total RNAs were extracted from CLN cells of mice infected with C. albicans and analyzed by RT-PCR using cytokine-specific primers. Equivalent loading of each sample was determined by the G3DPH message.

Article Snippet: Mice were injected intraperitoneally (i.p.) with 30 μg of rat anti-recombinant IL-4 (rIL-4) ( 29 ) (clone 11B11; PharMingen) or with the purified rat IgG1 matched isotype in 200 μl of PBS per mouse at days 1, 3, and 5 after oral infection with 10 8 yeast cells.

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Cellular and Cytokine Correlates of Mucosal Protection in Murine Model of Oral Candidiasis

doi:

Figure Lengend Snippet: IL-4, IL-12, and IFN-γ production by CLN cells stimulated in vitro. CLN cells from infected mice were stimulated with C. albicans antigen for 3 days, after which time the culture supernatants were assayed for cytokines by ELISA. Time zero represents uninfected mice. The results shown are means ± SEM for three to five mice. ∗, P < 0.05; ∗∗, P < 0.01 (values from BALB/c versus DBA/2 mice).

Article Snippet: Mice were injected intraperitoneally (i.p.) with 30 μg of rat anti-recombinant IL-4 (rIL-4) ( 29 ) (clone 11B11; PharMingen) or with the purified rat IgG1 matched isotype in 200 μl of PBS per mouse at days 1, 3, and 5 after oral infection with 10 8 yeast cells.

Techniques: In Vitro, Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Cellular and Cytokine Correlates of Mucosal Protection in Murine Model of Oral Candidiasis

doi:

Figure Lengend Snippet: Effect of treatment with an anti-IL-4 MAb on resistance to acute infection with C. albicans. BALB/c mice were injected i.p. with 30 μg of rat anti-IL-4 or with purified rat IgG1 matched isotype on days 1, 3, and 5 after challenge with yeast cells. On various days, the number of yeasts in the oral cavity was determined and the results were expressed as mean CFU ± SEM for three to five mice. ∗, P < 0.05.

Article Snippet: Mice were injected intraperitoneally (i.p.) with 30 μg of rat anti-recombinant IL-4 (rIL-4) ( 29 ) (clone 11B11; PharMingen) or with the purified rat IgG1 matched isotype in 200 μl of PBS per mouse at days 1, 3, and 5 after oral infection with 10 8 yeast cells.

Techniques: Infection, Injection, Purification

Journal:

Article Title: Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4

doi:

Figure Lengend Snippet: Borreliacidal antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi and treated with 0.01 (horizontal striped bar), 0.1 (checkered bar), or 1.0 (vertically striped bar) μg of rIL-4 10 min or 4 days (cross-hatched bar) (1 μg of rIL-4) after cultivation. Control cultures (solid bar) were treated with an equivalent volume of sterile PBS. Titers of borreliacidal antibody did not vary by more than 1 dilution in replicate assays. In most cases (90%) the same titer was obtained.

Article Snippet: In other experiments, recombinant IL-4 (rIL-4) or rat anti-murine IL-4 (R&D Systems, Minneapolis, Minn.) at quantities ranging from 0.01 to 1.0 μg was added to cultures of immune lymph node cells, macrophage, and B. burgdorferi at 10 min and 4 days of incubation.

Techniques: Cell Culture, Sterility

Journal:

Article Title: Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4

doi:

Figure Lengend Snippet: Borreliacidal antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi and treated with anti-murine IL-4 10 min (checkered bar) or 4 days (cross-hatched bar) after cultivation. Control cultures (solid bar) were treated with a non-isotype-specific antibody. Titers of borreliacidal antibody did not vary by more than one dilution in replicate assays. In most cases (90%) the same titer was obtained.

Article Snippet: In other experiments, recombinant IL-4 (rIL-4) or rat anti-murine IL-4 (R&D Systems, Minneapolis, Minn.) at quantities ranging from 0.01 to 1.0 μg was added to cultures of immune lymph node cells, macrophage, and B. burgdorferi at 10 min and 4 days of incubation.

Techniques: Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Behavior of  Visceral Leishmania donovani in an Experimentally Induced T Helper Cell 2 (Th2)-Associated Response Model

doi:

Figure Lengend Snippet: Preliminary experiments to determine the course of visceral infection in five different populations of mice. ( A ) BALB/c mice not treated (•) or injected once with IL-4/anti–IL-4 complex one day before (day −1) L. donovani challenge (○). ( B ) BALB/c mice not treated (•) or injected with IL-4/anti–IL-4 starting on day −1 and twice per week for the first four weeks after challenge (○). ( C ) Control 129/Sv/Ev (•) and 129/Sv/PEP IL-4 transgenic mice (○). ( D ) BALB/c mice treated three times per week for 2 wk with normal sheep IgG (•) or sheep anti–IL-12 IgG (○) starting 2 h after challenge. ( E ) BALB/c mice challenged with 10 7 (•) or 2 × 10 7 (○) amastigotes. In mice challenged with 2 × 10 8 amastigotes, week 8 LDU were 761 ± 61 ( n = 4 mice). Results in A–E indicate mean values (SE <16% in each case) from the single experiment performed with 4–5 mice per group. Note different vertical axis scale in B and C.

Article Snippet: 250 μg of murine recombinant IL-4 (rIL-4; lot 941-90-1; 10 8 U/ml, DNAX) was mixed in saline with 2.5 mg of rat anti–murine IL-4 mAb (11B.11; 10 mg/ml; DNAX) , and after 2 h on ice, 0.1 ml containing 10 μg of IL-4/100 μg of anti–IL-4 was injected intraperitoneally.

Techniques: Infection, Injection, Transgenic Assay

Journal: The Journal of Experimental Medicine

Article Title: Behavior of  Visceral Leishmania donovani in an Experimentally Induced T Helper Cell 2 (Th2)-Associated Response Model

doi:

Figure Lengend Snippet: Effect of anti–IL-4 or anti–IL-10 treatment on visceral infection of HKLPM-primed mice. BALB/c mice were pretreated once per week for 4 wk with HKLMP and then challenged with L. donovani . The day before challenge and once per week thereafter for 4 wk, mice received no further treatment ( filled bar ), injections of control antibody ( hatched bar ), or anti-cytokine mAb ( open bar ). Results are from two experiments, and indicate mean ± SEM LDU values for six to seven mice per group.

Article Snippet: 250 μg of murine recombinant IL-4 (rIL-4; lot 941-90-1; 10 8 U/ml, DNAX) was mixed in saline with 2.5 mg of rat anti–murine IL-4 mAb (11B.11; 10 mg/ml; DNAX) , and after 2 h on ice, 0.1 ml containing 10 μg of IL-4/100 μg of anti–IL-4 was injected intraperitoneally.

Techniques: Infection

Journal:

Article Title: Production of Borreliacidal Antibody to Outer Surface Protein A In Vitro and Modulation by Interleukin-4

doi:

Figure Lengend Snippet: Borreliacidal antibody response of lymph node cells obtained from 17-day-vaccinated mice cultured with macrophages and B. burgdorferi and treated with anti-murine IL-4 10 min (checkered bar) or 4 days (cross-hatched bar) after cultivation. Control cultures (solid bar) were treated with a non-isotype-specific antibody. Titers of borreliacidal antibody did not vary by more than one dilution in replicate assays. In most cases (90%) the same titer was obtained.

Article Snippet: In other experiments, recombinant IL-4 (rIL-4) or rat anti-murine IL-4 (R&D Systems, Minneapolis, Minn.) at quantities ranging from 0.01 to 1.0 μg was added to cultures of immune lymph node cells, macrophage, and B. burgdorferi at 10 min and 4 days of incubation.

Techniques: Cell Culture