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. 2010 Aug 6;285(32):24805-14.
doi: 10.1074/jbc.M110.142885. Epub 2010 Jun 4.

Different roles of G protein subunits beta1 and beta2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils

Yong Zhang  1 , Wenwen Tang, Matthew C Jones, Wenwen Xu, Stephanie Halene, Dianqing Wu
Affiliations

Different roles of G protein subunits beta1 and beta2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils

Yong Zhang et al. J Biol Chem. .

Abstract

Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gbeta(1) and/or Gbeta(2) in mouse neutrophils. Knockdown of Gbeta(2) appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gbeta(1) had no significant effect. However, knockdown of both Gbeta(1) and Gbeta(2) led to significant reduction in motility and responsiveness. In addition, knockdown of Gbeta(1) but not Gbeta(2) inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gbeta(1) and Gbeta(2) in the regulation of neutrophil functions.

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Figures

FIGURE 1.
FIGURE 1.
Suppression of Gβ2 expression in neutrophils using the LTR-YFP-shGβ2 vector. A, schematic representation of the LTR-YFP-shRNA vector. Gene-specific shRNA is embedded in the transcript of human miR30. To facilitate the selection, the vector also contains the YFP marker gene driven by the LTR promoter and the puromycin resistance gene regulated by the internal ribosome entry site sequence. B, Gβ expression in mouse neutrophils. Total RNAs were prepared from isolated bone marrow neutrophils and analyzed by the Affymetrix cDNA expression microarray system. The expression level of five Gβ isoforms was shown as the arbitrary unit (AU) of the microarray signal. C, neutrophils from non-transplanted mice or mice transplanted with cells infected with LTR-YFP-shGβ2 viruses were isolated and analyzed by a flow cytometer prior to sorting. D, flow cytometric analysis of sorted neutrophils from C. E, Western analysis of sorted YFP-positive neutrophils from D. Neutrophils were stimulated with or without 4 μm fMLP for 1 min, and cell lysates were separated by SDS-PAGE and detected with the indicated antibodies. NT, non-transplanted; LshGβ2, LTR-YFP-shGβ2; pS473, Ser(P)-473.
FIGURE 2.
FIGURE 2.
Efficient YFP expression and Gβ2 knockdown by the CMV-driven vector in NIH 3T3 cells. A, schematic diagram of the CMV-YFP-shRNA vector. YFP and shRNA are driven by the CMV promoter with a β-globin intron. B, NIH 3T3 cells were infected with viruses as indicated in the figure at a multiplicity of infection of 10 and observed under a fluorescence microscope with a 20× objective 5 days after infection. C, flow cytometric analysis of cells prepared as described in B. D, cells prepared as described in B were analyzed by Western blotting with the indicated antibodies.
FIGURE 3.
FIGURE 3.
Suppression of Gβ2 expression in primary mouse neutrophils using the CMV-YFP-shGβ2 vector. A, neutrophils were isolated from non-transplanted mice or mice transplanted with virus-infected bone marrow cells and analyzed by a flow cytometer. B, flow cytometric analysis of sorted YFP-positive neutrophils. C, sorted YFP-positive neutrophils were stimulated with or without 4 μm fMLP for 1 min, followed by Western analysis. NT, non-transplanted; shGβ2, CMV-YFP-shGβ2; shLuc, CMV-YFP-shLuc.
FIGURE 4.
FIGURE 4.
Suppression of Gβ1 expression or Gβ1 and Gβ2 expression in primary mouse neutrophils. A, flow cytometric analysis of sorted YFP-positive neutrophils from mice infected with CMV-YFP-shGβ1. B, sorted YFP-positive neutrophils were stimulated with or without 4 μm fMLP for 1 min, followed by Western analysis. NT, non-transplanted; shGβ1−, YFP-negative neutrophils sorted from mice infected with CMV-YFP-shGβ1 virus; shGβ1+, YFP-positive neutrophils sorted from mice infected with CMV-YFP-shGβ1 virus; shLuc+, YFP-positive neutrophils sorted from mice infected with CMV-YFP-shLuc. C, schematic diagram of the CMV-YFP-shRNA1-shRNA2 vector. D, flow cytometric analysis of sorted YFP-positive neutrophils from mice infected with CMV-YFP-shGβ1-shGβ2 virus. E, sorted YFP-positive neutrophils were stimulated with or without 4 μm fMLP for 1 min, followed by Western analysis. shGβ1,Gβ2−, YFP-negative neutrophils sorted from mice infected with CMV-YFP-shGβ1-shGβ2 virus; shGβIGβ2+, YFP positive neutrophils sorted from mice infected with CMV-YFP-shGβ1-shGβ2 virus; shLuc+, YFP-positive neutrophils sorted from mice infected with CMV-YFP-shLuc.
FIGURE 5.
FIGURE 5.
Chemotaxis assay for neutrophils from transplanted mice. Neutrophils isolated from transplanted mice were assayed for their chemotactic responses to an fMLP gradient in a Dunn chamber. As described under “Materials and Methods,” neutrophils were stimulated by fMLP and observed under a time lapse video microscope to monitor the migration of neutrophils using the Metamorph software. The directionality (A) and motility (B) of neutrophils infected with the indicated virus were determined in YFP-positive and YFP-negative fractions. The percentages of motile YFP+ cells relative to motile YFP− neutrophils are shown in C. *, p < 0.05 (paired Student's t test) between the YFP-positive and YFP-negative group. Error bars, S.E.
FIGURE 6.
FIGURE 6.
In vivo neutrophil infiltration assay for transplanted mice. A, mice transduced with the indicated virus were injected with 5 mg of carrageenan into their air pouch to induce the inflammation. 4 h later, cells from blood (top) or pouch exudates (bottom) were stained with APC-CD11b and PerCP-Ly-6G antibodies, and neutrophils (CD11b+Ly-6G+ cells) were gated out to analyze their YFP expression. YFP-negative and YFP-positive neutrophils are shown in the contour plots. B, the ratio between YFP-positive and YFP-negative neutrophils in the pouch exudates was normalized with the YFP-positive and YFP-negative neutrophil ratio in the blood in the same animal. *, p < 0.05; **, p < 0.01 (Student's t test) compared with the shLuc group. Error bars, S.E.
FIGURE 7.
FIGURE 7.
Phagocytosis and bacterial killing assay for neutrophils from transplanted mice. A, C, E, and G, mouse bone marrow cells isolated from the indicated mice transplanted with cells infected with shLuc (A and B), shGβ1 (C and D), shGβ2 (E and F), or shGβ1-β2 (G and H) were incubated at 37 °C in the presence of bacteria expressing red fluorescence at a ratio of 1:10 for 15 min. Unbound and non-internalized bacteria were then washed away, and red fluorescence intensity in neutrophils (Gr1hi cells) at this time point was used as a measurement of phagocytosis. B, D, F, and H, following bacterial loading, cells were incubated for the indicated time periods, and then fluorescence was measured by flow cytometry. The geometric mean of the red fluorescence between YFP-positive and YFP-negative Gr1hi neutrophils was plotted with the time in the indicated groups. *, p < 0.05 (paired Student's t test) between the YFP-positive and YFP-negative group. Error bars, S.E.
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