Figure 2
Figure 2. Siglec-9–positive leukocytes bind to vessels using VAP-1. (A) Expression of Siglec-9 on granulocytes used for ex vivo binding assays. Fluorescence-activated cell sorter histograms of CD66b and Siglec-9 expression are shown with and without incubation in FMLP-containing medium. Histograms with the negative control antibody are black. (B) Combined results of the induction experiments (mean ± SEM, n = 4) with FMLP, LPS, and TNF-α (values indicated/mL). (C) Expression of human VAP-1 (hVAP-1) is detected with biotinylated Jg.2-10 (red, left panel). Expression of PV-1–positive vessels in mesenteric lymph node vasculature of KOTG is detected by Meca-32 antibody (green g middle panel). The arrows indicate high endothelial venules. The merge of the panels is on the right. Insets: Stainings with a negative control antibody. Scale bar represents 50 μm. The images were taken with Olympus BX60 microscope using 40×/0.75 Ph2 objective and Olympus DP71 camera. The software was Cell D 2.6. (D) Ex vivo frozen section binding assays were used to analyze granulocyte binding to vessels in mesenteric lymph nodes obtained from VAP-1 KO and VAP-1 KOTG mice. The function of Siglec-9 was blocked by incubating the cells with anti–Siglec-9 antibody before the assay. (E) Granulocyte binding to inflamed synovial vessels. The granulocytes were pretreated with TNF-α and with anti–Siglec-9 and control antibodies as indicated. (D-E) The results are shown as percentage of control binding (number of KOTG vessel-bound or synovial vessel-bound granulocytes incubated with a nonblocking control mAb is defined as 100%; mean ± SEM). (F-H) Intravital analyses. Human granulocytes were fluorescently labeled with TAMRA and pretreated either with control antibody or anti–Siglec-9 mAb. Subsequently, the cells were injected into VAP-1 KOTG mice, and their interaction with the inflamed vessel wall was analyzed using intravital microscopy. (F) The graph indicates the percentage of rolling cells, calculated from the total number of cells appearing during the observation period. (G) The plots show the velocity of rolling cells. Each dot represents the mean rolling velocity of a single cell. (H) The graph represents the percentage of the cells that arrest on the venular wall for ≥ 30 seconds, calculated from the total number of rolling cells. In all 3 graphs, the horizontal lines indicate mean values. The number of mice/venules/PMN bolus injections were 3, 6, and 10 for control mAb and 3, 8, and 11 for anti–Siglec-9 mAb (F-H). *P < .05. **P < .01. ***P < .001. SSC-H indicates side scatter; FSC-H, forward scatter; and MFI, mean fluorescence intensity after subtracting the MFI obtained from the stainings with the negative control antibody.

Siglec-9–positive leukocytes bind to vessels using VAP-1. (A) Expression of Siglec-9 on granulocytes used for ex vivo binding assays. Fluorescence-activated cell sorter histograms of CD66b and Siglec-9 expression are shown with and without incubation in FMLP-containing medium. Histograms with the negative control antibody are black. (B) Combined results of the induction experiments (mean ± SEM, n = 4) with FMLP, LPS, and TNF-α (values indicated/mL). (C) Expression of human VAP-1 (hVAP-1) is detected with biotinylated Jg.2-10 (red, left panel). Expression of PV-1–positive vessels in mesenteric lymph node vasculature of KOTG is detected by Meca-32 antibody (green g middle panel). The arrows indicate high endothelial venules. The merge of the panels is on the right. Insets: Stainings with a negative control antibody. Scale bar represents 50 μm. The images were taken with Olympus BX60 microscope using 40×/0.75 Ph2 objective and Olympus DP71 camera. The software was Cell D 2.6. (D) Ex vivo frozen section binding assays were used to analyze granulocyte binding to vessels in mesenteric lymph nodes obtained from VAP-1 KO and VAP-1 KOTG mice. The function of Siglec-9 was blocked by incubating the cells with anti–Siglec-9 antibody before the assay. (E) Granulocyte binding to inflamed synovial vessels. The granulocytes were pretreated with TNF-α and with anti–Siglec-9 and control antibodies as indicated. (D-E) The results are shown as percentage of control binding (number of KOTG vessel-bound or synovial vessel-bound granulocytes incubated with a nonblocking control mAb is defined as 100%; mean ± SEM). (F-H) Intravital analyses. Human granulocytes were fluorescently labeled with TAMRA and pretreated either with control antibody or anti–Siglec-9 mAb. Subsequently, the cells were injected into VAP-1 KOTG mice, and their interaction with the inflamed vessel wall was analyzed using intravital microscopy. (F) The graph indicates the percentage of rolling cells, calculated from the total number of cells appearing during the observation period. (G) The plots show the velocity of rolling cells. Each dot represents the mean rolling velocity of a single cell. (H) The graph represents the percentage of the cells that arrest on the venular wall for ≥ 30 seconds, calculated from the total number of rolling cells. In all 3 graphs, the horizontal lines indicate mean values. The number of mice/venules/PMN bolus injections were 3, 6, and 10 for control mAb and 3, 8, and 11 for anti–Siglec-9 mAb (F-H). *P < .05. **P < .01. ***P < .001. SSC-H indicates side scatter; FSC-H, forward scatter; and MFI, mean fluorescence intensity after subtracting the MFI obtained from the stainings with the negative control antibody.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal