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. 2014 May 8;123(19):3027-36.
doi: 10.1182/blood-2013-06-507582. Epub 2014 Feb 26.

SRF is required for neutrophil migration in response to inflammation

Ashley Taylor  1 , Wenwen Tang, Emanuela M Bruscia, Ping-Xia Zhang, Aiping Lin, Peter Gaines, Dianqing Wu, Stephanie Halene
Affiliations

SRF is required for neutrophil migration in response to inflammation

Ashley Taylor et al. Blood. .

Abstract

Serum response factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of SRF loss in neutrophils, we crossed Srf(fl/fl) mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in Srf knockout (KO) neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to N-formyl-methionine-leucine-phenylalanine, resulting in significantly disrupted cytoskeletal remodeling. Srf KO neutrophils fail to migrate to sites of inflammation in vivo and along chemokine gradients in vitro. Polarization in response to cytokine stimuli is absent and Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in cellular adhesion, and although integrin expression levels are maintained with loss of SRF, integrin activation and trafficking are disrupted. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, underscoring an essential function for SRF and its pathway in health and disease.

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Figures

Figure 1
Figure 1
White blood cell counts and differential in Srf WT and KO mice and efficiency of Srf deletion. (A) WBC and white cell differential in primary Vav-Cre/Srf WT and KO mice. PB from Vav-Cre/Srf WT and KO mice aged 3-12 days was obtained and WBC and WBC differential assessed on Hemavet. (B) qRT-PCR on neutrophils flow sorted based on Gr-1high 7/4high granularityhigh criteria from Vav-Cre/Srf WT and KO mice. Srf expression is successfully abrogated in Vav-Cre/Srf KO neutrophils (100-fold, P < .0005; n = 4). *P < .05, **P < .005, ***P < .0005.
Figure 2
Figure 2
Assessment of neutrophil recruitment in vivo secondary to LPS-induced inflammation in the lung in Srf WT and KO mice. BAL was performed in Srf WT and KO mice before and 4 and 24 hours after LPS nebulization. Total (A), neutrophil (B), and macrophage (C) cell numbers were determined in BAL. (Combined data from 2 independent experiments: 0 hours, n = 4; 4 hours, n = 5; 24 hours, n = 4; *P < .05; **P < .005; ***P < .0005). Macrophage and neutrophil percentages were determined on Wright Giemsa–stained cytospins and by flow cytometry. (D-F) Migration of Srf WT and KO neutrophils in vitro in a transwell assay and chemokine receptor expression. Srf WT and KO neutrophils were allowed to migrate across 3-µm pore membrane toward an fMLP (D) and CXCL1 (E) gradient at indicated concentrations. Representative experiments performed in triplicate of at least 3 independent experiments (**P < .005; ***P < .0005). mRNA expression of chemokine receptors was assessed in Srf WT and KO neutrophils (F).
Figure 3
Figure 3
In vivo peritonitis model. (A) Peritonitis was induced in WT recipients and Srf WT, and KO neutrophils labeled with membrane dyes of 2 different colors were simultaneously injected intravenously and allowed to localize to tissues. PB, BM, and lavage fluid were harvested and percentages of donor WT vs KO neutrophils determined by flow cytometry detecting differential membrane staining (B-C). The experiment shown in panel B is representative of 4 independent experiments with n = 4 recipient mice of WT and KO neutrophils from 2 donors per experiment. Membrane dyes were alternated for WT and KO cells; **P < .005. (C) Primary flow data from 1 recipient showing donor KO and WT neutrophil distribution.
Figure 4
Figure 4
Srf KO neutrophils lack polarization with decreased f-actin polymerization when activated with fMLP. Srf WT and KO neutrophils were allowed to attach to poly-l-lysine–coated coverslips and stimulated with fMLP for 2 minutes. (A) F-actin was stained with phalloidin (middle) in Cre-expressing neutrophils marked by YFP (bottom); merge with DAPI is shown in the top panels. Quantification of polarized cells from 3 independent experiments; Srf WT, n = 71; Srf KO, n = 91 (B). G-actin (C) and F-actin (D) were assessed at 0, 30, 60, and 90 seconds after stimulation with fMLP by staining with DNAse I (G-actin) and phalloidin (F-actin) and mean fluorescence intensity (MFI) determined by flow cytometry. Representative of 3 independent experiments performed in triplicate.
Figure 5
Figure 5
Integrin homeostasis in Srf WT and KO neutrophils. (A) Expression of integrin CD11b, CD18, and CD11a on the cell surface of Srf WT and KO neutrophils by flow cytometry. Srf WT and KO neutrophils were incubated with fluorescently labeled ICAM-1 (B) and stimulated with fMLP for 0, 2, and 5 minutes; (C) CD11b surface expression was determined at the same time. (Representative experiment of 3 independent experiments performed in triplicate; **P < .005, ***P < .0005.) (D) Srf WT and KO neutrophils were stimulated with vehicle or fMLP for 15 minutes at 37°C and lysates probed for Itgam, Erk, and P-Erk, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. Lysates are from 1 experiment; vertical lines have been inserted to indicate a repositioned gel lane. Neg ctrl, negative control.
Figure 6
Figure 6
Integrin localization in Srf WT and KO neutrophils. Srf WT (left) and KO (right) neutrophils were (A) allowed to adhere to glass coverslips, stained with CD11b fluorescein isothiocyanate, and then stimulated with fMLP for 0 (none) and 15 (fMLP) minutes and stained with wheat germ agglutinin antibody as a membrane stain (without permeabilization) and DAPI (A). (B-C) Srf WT and KO neutrophils were allowed to adhere to glass coverslips, stimulated with fMLP for 0 (none) and 15 (fMLP) minutes and stained with antibodies against CD11b and Clathrin (B) and CD11b and Kindlin (C). (A-B) The top 3 panels show single sections of the Z-stack; the bottom panel shows a merge of the Z-stack. (C) Three sequential Z-stack sections for each image. DAPI stains nuclei. Scale bar = 6 µm; >, leading edge; *, trailing edge.
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