Figure 3
Figure 3. Knockdown of Gnb isoforms in primary neutrophils significantly abolishes chemokine-induced arrest in vivo and neutrophil migration in vitro. Retrovirally transduced hematopoietic stem cells with shRNAs against Gnb1, Gnb2, Gnb4, and Gnb5 or with a scrambled construct were transplanted into lethally irradiated WT recipients. (A) Mixed chimeric mice were exposed to nebulized saline or LPS to induce lung injury. Data indicate number of neutrophils in the BAL 24 hours after inhalation of NaCl (left panel) or LPS (right panel) (n = 3). *P < .05 vs all other groups. (B) Mixed chimeric mice were used to investigate CXCL1-induced arrest in the cremaster muscle (n = 3). *P < .05 vs all other groups. (C) Purified bone marrow–derived neutrophils were used for an ICAM-1-binding assay. During flow cytometry acquisition, cells were gated into GFP-positive (shRNA-containing) and GFP-negative (WT) cell populations and analyzed for CXCL1-induced increase of bound APC-labeled ICAM-1. Data represent MFI of APC in nontransduced and transduced cells, both unstimulated and stimulated (n = 4 experiments). *P < .05 vs all other groups. (D) Isolated PMNs from mixed chimeric mice were left untreated or were stimulated with CXCL1, fixed with paraformaldehyde (4%), and stained with anti-CD11a and AF488-labeled secondary antibody to visualize clustered LFA-1 (n = 3 experiments). *P < .05 vs all other groups. (E) Purified bone marrow–derived neutrophils were left unstimulated or incubated with CXCL1 (100 ng/mL, 3 minutes, 37°C), fixed, and permeabilized to stain intracellular p-p38 MAPK. MFI of bound AF647-labeled p38 MAPK antibody was analyzed by using flow cytometry gating into GFP-positive and GFP-negative populations (n = 3 experiments). *P < .05 vs all other groups. (F-H) Chemotactic migration of purified bone marrow–derived neutrophils of mixed chimeric mice (n = 3 experiments; >40 cells). *P < .05 vs all groups. (F) Migration directness and (G) migration velocity toward the applied CXCL1 gradient in the ibidi μ-Slide. (H) Representative migration plots of cells transduced with a scrambled construct or with an shRNA against 1 Gnb isoform. Arrow indicates applied CXCL1 gradient. no c., no construct; n.s., not significant.

Knockdown of Gnb isoforms in primary neutrophils significantly abolishes chemokine-induced arrest in vivo and neutrophil migration in vitro. Retrovirally transduced hematopoietic stem cells with shRNAs against Gnb1, Gnb2, Gnb4, and Gnb5 or with a scrambled construct were transplanted into lethally irradiated WT recipients. (A) Mixed chimeric mice were exposed to nebulized saline or LPS to induce lung injury. Data indicate number of neutrophils in the BAL 24 hours after inhalation of NaCl (left panel) or LPS (right panel) (n = 3). *P < .05 vs all other groups. (B) Mixed chimeric mice were used to investigate CXCL1-induced arrest in the cremaster muscle (n = 3). *P < .05 vs all other groups. (C) Purified bone marrow–derived neutrophils were used for an ICAM-1-binding assay. During flow cytometry acquisition, cells were gated into GFP-positive (shRNA-containing) and GFP-negative (WT) cell populations and analyzed for CXCL1-induced increase of bound APC-labeled ICAM-1. Data represent MFI of APC in nontransduced and transduced cells, both unstimulated and stimulated (n = 4 experiments). *P < .05 vs all other groups. (D) Isolated PMNs from mixed chimeric mice were left untreated or were stimulated with CXCL1, fixed with paraformaldehyde (4%), and stained with anti-CD11a and AF488-labeled secondary antibody to visualize clustered LFA-1 (n = 3 experiments). *P < .05 vs all other groups. (E) Purified bone marrow–derived neutrophils were left unstimulated or incubated with CXCL1 (100 ng/mL, 3 minutes, 37°C), fixed, and permeabilized to stain intracellular p-p38 MAPK. MFI of bound AF647-labeled p38 MAPK antibody was analyzed by using flow cytometry gating into GFP-positive and GFP-negative populations (n = 3 experiments). *P < .05 vs all other groups. (F-H) Chemotactic migration of purified bone marrow–derived neutrophils of mixed chimeric mice (n = 3 experiments; >40 cells). *P < .05 vs all groups. (F) Migration directness and (G) migration velocity toward the applied CXCL1 gradient in the ibidi μ-Slide. (H) Representative migration plots of cells transduced with a scrambled construct or with an shRNA against 1 Gnb isoform. Arrow indicates applied CXCL1 gradient. no c., no construct; n.s., not significant.

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