Fig 1: Relationship between CX3CR1 gene expression levels and AAT deficiency and the concentration of circulating AAT polymers.CX3CR1 gene expression levels in peripheral blood mononuclear cells (PBMCs) related to alpha-1 antitrypsin-deficiency (AATD) and plasma concentrations of CX3CR1 ligand (CX3CL1) and Z-AAT polymer. (A)PBMCs were isolated from AATD subjects and non-AATD controls. CX3CR1 gene expression relative to HPRT1 housekeeping gene was determined by real-time PCR using Taqman gene expression assays. Measurements were carried out in duplicates. Data are presented as median (IQR) in boxplots, lines represent medians. Outliers are defined as data points located outside the whiskers. p-Value was calculated by Mann-Whitney U test. (B) Plasma levels of CX3CL1 in AATD (plasma available for n = 38 AATD) and non-AATD individuals measured by ELISA. Measurements were carried out in triplicates. Data are presented as median (IQR) in boxplots with whiskers. Outliers are defined as data points located outside the whiskers. (C) Negative correlation of CX3CR1 mRNA in PBMCs and plasma Z-AAT polymer levels from ZZ AATD individuals from graph (B). Pearson’s correlation test, r2 = −0.313, p=0.055, n = 38.Figure 1—source data 1.Source files, containing original data for Figure 1A and B, to document CX3CR1 expression (A), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals (B).
Fig 2: Naïve NK cells migrate towards IL-8 and Fractalkine but not to vMIP-II.Transwell migration assay was performed with the following recombinant proteins: rhIL-8 (A), rhFck (B) or rvMIP-II (C) placed in the bottom chamber and freshly isolated naïve NK cells were placed in the upper chamber. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). Rh - recombinant human. Rv - recombinant viral. *P<0.05. ***P<0.001. NS - not significant. Figure shows one representative experiment out of four performed.
Fig 3: vMIP-II blocks the migration of freshly isolated naïve NK cells to Fractalkine.(A) Freshly isolated naïve NK cells were double stained with Fck-Ig and with anti-CD56 mAb. The percentages of the various populations are indicated in the figure. (B) CX3CR1 expression on the transfectant 293T-CX3CR1 cells (black open histogram) or on 293T parental cells (filled grey histogram). (C) Binding of Fck-Ig (left histogram) or vMIP-II-Ig (right histogram) to 293T-CX3CR1 transfectant (black open histogram) or to the 293T parental cells (filled grey histogram). (D) 293T-CX3CR1 cells were incubated with (black empty histogram) and without (dark gray empty histogram) rvMIP-II for 1 hour in 4°C and then stained with Fck-Ig. The light gray filled histogram is the staining of Fck-Ig on the 293T parental cells. (E) Freshly isolated naïve NK cells were incubated at 4°C for 1 hour with and without the proteins indicated in the x axis. RhFck was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). **P<0.01. NS - not significant. Figure shows one representative experiment out of four performed.
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