Fig 1: Metoprolol directly inhibits neutrophil deleterious function through a ADRB1-blockade.(a) Effect of metoprolol on CXCL1-induced migration of fresh isolated primary neutrophils (Ly6G+) from WT or Adrb1-knockout (β1KO) mice. CXCL1-stimulated cells were incubated with vehicle, epinephrine (10 μM), metoprolol (10 μM) and epinephrine+metoprolol; n=4 independent experiments. NS, stands for non-significant. (b,c) Inhibitory effect of metoprolol on W-peptide-induced ROS production on fresh isolated primary neutrophils (Ly6G+) from WT or β1KO mice. Mean fluorescent intensity of Rho123 in Ly6G+ neutrophils after W-peptide stimulation. n=6 independent experiments; flow cytometry plots illustrate reduced expression of Rho123 in metoprolol-treated neutrophils. (d–f) Effect of metoprolol on limiting-thioglycolate-induced peritoneal infiltration on WT and β1KO mice. (e) Flow cytometry plots illustrating reduced infiltration of neutrophils (CD115neg; GR1+) in metoprolol-treated mice. Absolute neutrophils detected per ml of infiltrate at 16 h after thioglycolate injection in WT mice (n=7–9) or β1KO mice (n=5). (g) Effect of metoprolol on thioglycolate-induced neutrophil infiltration in the four BMT groups. Protocol scheme for thioglycolate-induced peritonitis assay after bone-marrow transplants (BMT) between WT and β1KO mice, evaluating the influence of the presence or absence of ADRB1 in circulating cells. Data are normalized to vehicle; n=4–9. (h) Protocol scheme for IR experiments in chimeric animals after BMT, evaluating the infarct-limiting effect of metoprolol in the presence or absence of ADRB1 in circulating cells. (i–q) Area at risk (AAR) and infarct size, as well as representative images of Evans blue and TTC staining in metoprolol-treated and vehicle-treated β1KO and chimeras (WT bone marrow transplanted into β1KO mice and reverse transplants). Infarct size is reduced by metoprolol only when circulating cells express β1-adrenergic receptor; n=9 (i,j), n=8 (l,m), n=5–6 (o,p). Data are means±s.e.m. *P<0.05; **P<0.01; ***P<0.001, determined by the nonparametric Wilcoxon–Mann–Whitney test or using the one-way ANOVA and Holm Sidak's post-hoc multiple comparisons method.
Fig 2: Local β2 integrin activation in mouse neutrophils arresting in response to CXCL1 in vivo(A) Induction of β2 extension (KIM127-DL550, red) and high-affinity (mAb24-AF488, green) conformations in mouse neutrophils in vivo. Fluorescence images of adherent neutrophils in mouse cremaster muscle venules of hITGB2 KI mice before (left panel) and after (right panel) injection of 300 ng CXCL1 via a carotid artery catheter. White arrows indicate arrested neutrophils. Neutrophils are identified by expression of Ly6G-AF647 (magenta). Horizontal black arrow indicates the direction of blood flow (from right to left). The asterisk indicates a rolling neutrophil. Scale bars, 20 μm.(B) Eight arrested neutrophils in vivo (two mice), showing distribution of activated β2 integrins.(C) MFI of mAb24 binding and KIM127 binding before (n = 6 neutrophils) and after (n = 8 neutrophils) CXCL1 treatment.(D) Upper panel: a line (1 pixel wide using the plot profile function in ImageJ software) was drawn along the cell top, front, bottom, and rear of each neutrophil based on Ly6G labeling. Profiles of mAb24 and KIM127 fluorescence intensities were measured as a function of location. Distance normalized to the circumference of each cell (0–1). Lower panel: profile of mAb24 and KIM127 fluorescence intensities were normalized to average signal intensities. Colocalized signal is represented by the mAb24 signal multiplied by KIM127 signals (mAb24xKIM127, yellow traces).(E) Statistical comparison of mAb24 and KIM127 fluorescence intensities in the front and rear of neutrophils. Means ± SEM of 8 neutrophils are shown.(F) Distance around each circumference was normalized (0–1). Intensities for mAb24, KIM127, and the product KIM127 × mAb24 were normalized so that the average intensity was equal to 1 (horizontal line). Data were obtained from 8 neutrophils at 1–2 min after arrest. The normalized data points were binned into 40 intervals and plotted with the R package ggplot2. Point range bars show the range of each binned data point. Statistical analysis was performed for mAb24 × KIM127 by a one-sample t test with Benjamini-Hochberg correction, comparing the overall average (μ = 1) to each binned data point’s average.*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.
Fig 3: Breast tumor–induced signals reprogram BM neutrophils’ gene expression.(A) Scatterplot of the mRNA expression ln(mean expression + 1) between BM neutrophils derived from naïve versus MMTV-PyMT mice. Labeled genes are the top 50 differentially expressed genes (ranked by adjusted P value; generalized linear model tested with the quasilikelihood F test with Benjamini-Hochberg correction). Blue and red dots represent significantly (adjusted P value < 0.05 and log2FC > 1.0 or log2FC < −1.0) up-regulated genes and down-regulated genes in MMTV-PyMT neutrophils, respectively. (B) Heatmap of z-scored mRNA expression for the top 70 differentially expressed genes (ranked by adjusted P value < 0.05) that comprise the neutrophil reprogramming signature induced by tumor burden. Columns represent individual replicates. Hierarchical clustering with Euclidean distance produced the dendrogram. (C) qRT-PCR analysis of selected tumor-induced signature genes in BM neutrophils from naïve and MMTV-PyMT mice. Bar graphs indicate means ± SEM of 2−ddCT mRNA expression normalized to naïve neutrophils. One-tailed paired t test used. Three mice per genotype were used. (D) Western blot showing TSPO and IFITM1 from BM neutrophils of naïve and MMTV-PyMT mice. (E) Heatmap depicting the mean inferred TF activity scores for individual replicate samples from naïve versus PyMT BM neutrophils. Inference scores were calculated using a ULM in individual cells and then averaging the scores per sample. Arrows indicate TFs mentioned in the text. TFs whose average activity has the highest degree of variance between genotypes are shown. Pearson correlation between TF expression and activity is shown. (F) Heatmap of z-scored mRNA expression for the top 10 genes (highest Moran’s I statistic) enriched in N5_N-specific (left) or N5_P-specific (right) Monocle3-derived gene modules in clusters N3, N4, and N5_N/P. Columns represent N clusters. (G) Transwell migration of BM neutrophils across a 3-μm membrane barrier without (spontaneous) and with 30 nM the CXCL1 chemokine in the lower chamber. Bar graphs indicate means ± SEM of transmigrated neutrophils. Two-tailed t test used.
Supplier Page from R&D Systems, a Bio-Techne Brand for CXCL1/GRO alpha/KC/CINC-1 aa 20-96 Protein
Available conjugates: Sizes Available: 10 ug (also 50 ug)