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1.
Figure 3

Figure 3. CXCL5 inhibits chemokine scavenging in whole blood in vitro and in vivo. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

The plasma concentrations of CXCL1 (A), CXCL2 (B) and CXCL5 (C) were measured by ELISA after incubation of the citrated blood from WT and Cxcl5−/− mice (n=3 mice/group) with recombinant CXCL1, CXCL2 and CXCL5 respectively for 15 minutes. The plasma concentrations of CXCL1 (D), CXCL2 (E) and CXCL5 (F) in the plasma of WT and Cxcl5−/− mice were measured at different time points after intravenous injection of recombinant CXCL1, CXCL2 and CXCL5 respectively. Data are representative of two independent experiments. N.D: not detected. *P<0.05; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.
2.
Figure 7

Figure 7. The origin of CXCL5 in both LPS inhalation and severe E.coli pneumonia model. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

WT and Cxcl5−/− mice were reconstituted with BM from WT and Cxcl5−/− mice respectively, after 8 weeks, the chimeric mice were exposed to 0.3 mg/ml LPS for 30 minutes (A), or intratracheally challenged with 107 CFU E.coli (n=3 mice/group) (B,C). At 8 hours after challenge, the BALF (A, B) and plasma samples (C) were prepared and CXCL5 measured by ELISA. WW: BM from WT donor mice into WT recipient mice; WK: BM from WT donor mice into Cxcl5−/− recipient mice; KW: BM from Cxcl5−/− donor mice into WT recipient mice; KK: BM from Cxcl5−/− donor mice into Cxcl5−/− recipient mice. Data are representative of two independent experiments. N.D: not detected.

Junjie Mei, et al. Immunity. ;33(1):106-117.
3.
Figure 6

Figure 6. CXCL5 is required for pulmonary immune responses to LPS inhalation. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

WT and Cxcl5−/− mice (n=3 mice/group) were exposed to nebulized LPS (0.3 mg/ml) for 30 minutes, at different time point, white blood cells (WBC) (A) and neutrophils (B) were counted in the BALF. The lung MPO activities (C) were measured from lung homogenate. The protein amounts of TNF-α (D), IL-6 (E), CXCL5 (F), CXCL1 (G), CXCL2 (H) and CXCL15 (I) in the BALF were measured by ELISA. At 4 and 24 hours after LPS inhalation, the blood was drawn from inferior vena cava after injection of 3.2% sodium citrate and the plasma concentrations of CXCL5 (J), CXCL1 (K), CXCL2 (L) and CXCL15 (M) were measured by ELISA. Data are representative of two independent experiments. N.D: not detected. *P<0.05; **P<0.01; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.
4.
Figure 2

Figure 2. Plasma chemokines, chemokine gradients, and CXCR2 desensitization upon E.coli challenge. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

WT and Cxcl5−/− mice (n=4 mice/group) were i.t. inoculated with 107 CFU E.coli, at different time points, the blood was drawn from vena cava after injection of 3.2% sodium citrate. The plasma amounts of CXCL5, CXCL1 and CXCL2 were measured upon E.coli challenge (A,B,C) by ELISA. The ratio of BALF to plasma CXCL1 (D) and CXCL2 (E) were calculated with the mean value of chemokine concentrations from Figure 1G–H and Figure 2B–C. The gating of Gr-1+ cell in the BM (F) and the representative calcium responses (G) of BM Gr-1+ cells of naïve WT mice (red line), E.coli-challenged (24 hours after challenge) WT (blue line) and Cxcl5−/− mice (green line)(n=3 mice/group) to 10 nM CXCL1 are shown. The BM cells were stained with APC-conjugated Gr-1 antibody and labeled with indo-1 AM. Data are representative of two independent experiments. N.D: not detected. **P<0.01; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.
5.
Figure 4

Figure 4. CXCL5 regulates plasma chemokines at least in part through homeostatic and high-affinity binding with erythrocyte DARC. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

The plasma concentrations of CXCL5 (A) and CXCL1 (B) were measured by ELISA after incubation of the citrated blood from WT and Darc−/− mice (n=4 mice/group) with recombinant CXCL5 or CXCL1 respectively for 15 minutes. The amounts of plasma and erythrocyte DARC-binding CXCL1, CXCL2 and CXCL5 in blood of naïve WT mice (n=3 mice/group) were measured by ELISA (C). The amounts of plasma CXCL1 and erythrocyte DARC-binding CXCL1 were also measured by ELISA from the citrated blood 15 minutes after intravenous injection of 1µg recombinant CXCL1 into WT and Cxcl5−/− mice (n=3 mice/group) (D). Competitive binding assay using 125I-hGro-α (CXCL1) (E) or 125I-hCCL2 (G) for purified murine erythrocytes from WT (E,G) and Darc−/− (G, CXCL5-70 Darc−/−) mice with various concentrations of cold ligands CXCL5-70, CXCL1, CXCL2, CXCL5-93 and unlabeled hGro-α (CXCL1) or hCCL2 were performed. The Ki (equilibrium dissociation constant) values (F, H) for cold competitors (n=4 times) were calculated and show distinct binding affinities for red cell DARC within the ELR+ CXC chemokines family (p<0.05 for all ligands comparisons). Darc−/− red cells do not bind appreciably radiolabeled ligands (G). The percentages of erythrocyte DARC-binding CXCL1 in blood (I) were calculated after measurement of plasma and erythrocyte DARC-binding CXCL1 in blood of WT and Cxcl5−/− mice (n=3 mice/group) at different time points upon 107 CFU E.coli i.t. challenge. N.D: not detected. *P<0.05; **P<0.01; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.
6.
Figure 1

Figure 1. The pulmonary immune responses, lung injury and host defense upon E.coli intratracheal challenge. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

White blood cells (A) and neutrophils (B) were counted in the BALF of WT and Cxcl5−/− mice (n>=5 mice/group) at 8 and 24 hour point after 107 CFU E.coli (intratracheal (i.t.) inoculation. The lung myeloperoxidase activities (C) were measured from lung homogenate. The lung bacterial burden (D) was measured in the lung homogenate by serial dilution on MacConkey plates. The mortality rate of mice (E) was monitored within 24 hours after inoculation, and the fractions above the columns represent the number of dead mice among the number of mice inoculated. The lung wet/dry ratios (F) were measured after drying the whole lung in 80°C oven for 48 hours. (n>=5 mice/group). Concentrations of CXCL1 (G), CXCL2 (H), CXCL5 (I), TNF-α (J) and IL-6 (K) in the BALF were measured by ELISA. Control mice were i.t. challenged with sterile saline. Data are representative of two independent experiments. Representative haematoxylin & eosin staining (L) of fixed lung samples at 24 hours of WT and Cxcl5−/− mice (n=4 mice/group) is shown. The lungs were fixed with 10% neutral formalin. Arrows indicate E.coli rods. Size bar: 40 µm. N.D: not detected. *P<0.05; **P<0.01 ; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.
7.
Figure 5

Figure 5. Large amounts of preformed CXCL5 in platelets and platelet origin for homeostatic CXCL5 in blood. From: CXCL5 Regulates Chemokine Scavenging and Pulmonary Host Defense to Bacterial Infection.

Comparison of plasma concentrations of CXCL5 (A) by retro-orbital bleeding (with coagulation) and vena cava bleeding (by injection of sodium citrate, without coagulation) in WT mice (n=4 mice/group) upon 107 CFU E.coli i.t. challenge, measured by ELISA. The plasma concentrations of CXCL1 (B), CXCL2 (C) and CXCL15 (D) by retro-orbital bleeding from naïve WT mice and Cxcl5−/− mice (n=4 mice/group) were measured by ELISA. CXCL5, but not CXCL1, is detected in inactivated platelets of naïve WT mice by western blot (E), and both preformed and secreted CXCL5 by thrombin stimulation from platelet is mostly CXCL5-93 form (F). R: recombinant CXCL5-93 (9.8 kD) and CXCL5-70 (7.6 kD); Fr/th: the supernatant after separated platelets were snap-frozen and thawed twice; thrombin: the supernatant after separated WT platelets were stimulated with 1 U/ml thrombin for 10 minutes at 37°C;WT or Mu: lysate of inactivated platelets in WT or mutant Cxcl5−/− mice. WT and Cxcl5−/− mice were reconstituted with BM from WT and Cxcl5−/− mice respectively, after 8 weeks, the citrated blood will be prepared from the mice in each group (n=4 mice/group). The plasma and red cells were separated, then the plasma CXCL5 concentrations (G) and erythrocyte-binding CXCL5 (H) were measured by ELISA. After incubation of the citrated blood with recombinant CXCL5 for 15 minutes, the plasma concentrations of CXCL5 (I) were measured. WW: BM from WT donor mice into WT recipient mice; WK: BM from WT donor mice into Cxcl5−/− recipient mice; KW: BM from Cxcl5−/− donor mice into WT recipient mice; KK: BM from Cxcl5−/− donor mice into Cxcl5−/− recipient mice. The amounts of plasma CXCL5 and erythrocyte-binding CXCL5 in citrated blood of Mpl−/− mice (J), Fog-1ki/ki mice (K) and their respective WT control mice were measured by ELISA. The plasma concentrations of CXCL4 and CXCL7 were measured in the citrated blood of Mpl−/− mice (M), Fog-1ki/ki mice (N) and their respective WT control mice as well. Only the CXCL7-74 (β-thromboglobulin) form is detected in platelets by Western blot (L). R: recombinant CXCL7-74 a.a. (8.2 kD). Data are representative of two independent experiments. N.D: not detected. *P<0.05; **P<0.01; ***P<0.001.

Junjie Mei, et al. Immunity. ;33(1):106-117.

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