Journal: PLoS ONE

Article Title: Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4 + T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

doi: 10.1371/journal.pone.0014616

Figure Lengend Snippet: The differently expressed proteins identified based on single peptides in CD4 + T cells between active VKH patients and normal individuals.

Article Snippet: Membranes were incubated with antibodies at dilutions of 1∶1000 for both anti-human CD18 mAb (R&D systems, Minneapolis, MN) and anti-human AKNA mAb (Genway Biotech, San Diego, CA).

Techniques: Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4 + T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

doi: 10.1371/journal.pone.0014616

Figure Lengend Snippet: Antibodies were used at a dilution of 1∶1000 for the anti-human CD18 monoclonal antibody and anti-human AKNA monoclonal antibody. Proteins were detected using the Phototope-HRP Western blot detection system(A). The immunoreactive band intensities were quantitated and were presented as intensity volumes (vol%). The results showed that both CD18 and AKNA were significantly down-regulated in VKH patients as compared to normal controls (B). VKH: VKH patients, NC: normal controls.

Article Snippet: Membranes were incubated with antibodies at dilutions of 1∶1000 for both anti-human CD18 mAb (R&D systems, Minneapolis, MN) and anti-human AKNA mAb (Genway Biotech, San Diego, CA).

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

doi: 10.4049/jimmunol.167.3.1431

Figure Lengend Snippet: FIGURE 1. Binding of COS-7 transfectants to ICAM-1. A, Average adhesion to ICAM-1 of COS-7 cells transiently transfected with WT or mutant CD11a and CD18. The assay was performed in wells without ICAM-1 (BSA) and in wells with ICAM-1 and buffer alone (ICAM-1), isotype-matched control mAb 1B7 (Control), CD11a-specific blocking mAb TS1/22 (Block), and activating mAb 240Q (240Q). The adhesion assay is representative of three independent experiments. Mean OD values (expressed as a percentage of those obtained with capture Abs TS1/22 and TS1/18) and SDs from triplicate wells are shown. B, Average adhesion to ICAM-1 of COS-7 cells transiently transfected with WT and single mutant or double mutant CD11a and CD18. Experimental conditions were as de- scribed in A.

Article Snippet: Hybridoma cells producing anti-human CD11a mAb (TS1/22, IgG1), or anti-human CD18 mAb (TS1/18, IgG1) were obtained from American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Binding Assay, Transfection, Mutagenesis, Control, Blocking Assay, Cell Adhesion Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

doi: 10.4049/jimmunol.167.3.1431

Figure Lengend Snippet: FIGURE 2. Characterization of inhibitory CD11a mutants in Jb2.7 cells and binding to ICAM-1. A, Expression of LFA-1 on Jb2.7 clones trans- fected with WT or inhibitory LFA-1 mutants. Clones were stained with an anti-CD18 mAb (TS1/18) and analyzed by FACS to verify the expression levels of LFA-1. The open histogram represents untransfected Jb2.7 cells. Filled histograms represent CD11A transfected Jb2.7 clones. Matched WT

Article Snippet: Hybridoma cells producing anti-human CD11a mAb (TS1/22, IgG1), or anti-human CD18 mAb (TS1/18, IgG1) were obtained from American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Binding Assay, Expressing, Clone Assay, Staining, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cellular activation of leukocyte function-associated antigen-1 and its affinity are regulated at the I domain allosteric site.

doi: 10.4049/jimmunol.167.3.1431

Figure Lengend Snippet: FIGURE 3. Characterization of activating CD11a mutants in Jb2.7 cells and binding to ICAM-1. A, Expression of LFA-1 on Jb2.7 clones transfected with WT or activating LFA-1 mutants. Clones were stained with an anti-CD18 mAb (TS1/18) and analyzed by FACS to verify the expression levels of LFA-1. The open histogram represents untransfected Jb2.7 cells. Filled histo- grams represent CD11A-transfected Jb2.7 clones. Matched WT clones were selected based on equivalent or higher relative LFA-1 expression to negate any

Article Snippet: Hybridoma cells producing anti-human CD11a mAb (TS1/22, IgG1), or anti-human CD18 mAb (TS1/18, IgG1) were obtained from American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Binding Assay, Expressing, Clone Assay, Transfection, Staining

Journal:

Article Title: Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-?1 Generation and Monocyte Binding

doi:

Figure Lengend Snippet: Disruption of CD44-mediated U937 cell interaction with HA increases TGF-β1 promoter activity. U937 cells (0.3 × 106) pretreated with increasing concentration of blocking antibody to CD44, at 37°C for 60 minutes, were added to TGF-β1 promoter reporter construct-transfected HK-2 cells (αCD44) in the presence of the blocking antibody. In control experiments U937 cells (0.3 × 106) were added to transfected HK-2 cells in the absence of any blocking antibody (control) or in the presence of 5 μg/ml of irrelevant IgG1 antibody control (IgG). In an additional experiment U937 cells were pretreated with 5 μg/ml of anti-CD44 antibody and either soluble 400 ng/ml ICAM-1 (+ICAM) or 10 μg/ml anti-CD18 antibody (+CD18) for 1 hour at 37°C, before their addition to transfected HK-2 cells pretreated with blocking antibody to CD44. During the co-culture period, cells were incubated with the CD44 antibody and also either the soluble ICAM or anti-CD18 antibody. TGF-β1 promoter activity was expressed as the change in relative value comparing antibody-treated U937 cells to those not treated with antibody. Results represent mean ± SD; n = 6; *, P < 0.0005, compared to the serum-free control.

Article Snippet: Other reagents and sources were as follows: testicular hyaluronidase (H3884) (Sigma, Poole, UK), recombinant human soluble ICAM-1 (R&D Systems Europe Ltd.), anti-human CD44 blocking monoclonal antibody BU75 (Ancell Corp., USA), anti-human CD18 blocking monoclonal antibody (Ancell Corp.), anti-human anti-CD54 (ICAM-1) cross-linking monoclonal antibody 6.5 (clone RR 6.5; a gift from Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, NJ), and anti-mouse IgG (whole molecule) (Sigma).

Techniques: Activity Assay, Concentration Assay, Blocking Assay, Construct, Transfection, Co-Culture Assay, Incubation

Journal:

Article Title: Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-?1 Generation and Monocyte Binding

doi:

Figure Lengend Snippet: Inhibition of ICAM-1-CD18 interaction abrogates TGF-β1 generation after addition of U937 cells. A: Inhibition of TGF-β1 promoter activity by CD18-blocking antibody. U937 cells, either untreated (U937) or pretreated with 10 μg/ml of anti-CD18 antibody (+CD18) or irrelevant IgG1 control (+IgG) for 1 hour at 37°C were added to HK-2 cells transfected with the TGF-β1 promoter-luciferase construct. In the control experiment, serum-free medium alone was added to the transfected cells. TGF-β1 promoter activity was expressed as the relative change in value compared to the serum-free control. Results represent mean ± SD, n = 6. B: Inhibition of monocyte binding by sICAM: confirmation that inhibition of ICAM1-CD18 interaction reduced monocyte binding was demonstrated by addition of 1 × 106 51Ci chromium-labeled U937 cells under serum-free conditions for 1 hour at 37°C to confluent monolayers of HK-2 cells in the presence of an increasing concentration (0 to 800 ng/ml) soluble ICAM. Quantitation of bound radioactivity was performed as described in Materials and Methods and results represent mean ± SD of six individual experiments. C: Inhibition of TGF-β1 protein synthesis by blocking CD18. U937 cells, either untreated (stippled bars) or pretreated with 10 μg/ml of anti-CD18 antibody (solid bars) or irrelevant IgG1 control (cross-hatched bars) for 1 hour at 37°C were added to confluent monolayers of HK-2 cells and TGF-β1 quantified by ELISA at the indicated time points. Results represent mean ± SD, n = 5. D: Addition of U937 cells to the basolateral aspect of HK-2 cells increases TGF-β1 transcription. HK-2 cells were subcultured and seeded onto the bottom side of polycarbonate tissue culture inserts (pore size, 3.0 μm). In the inverted position the cells were allowed to attach for 4 hours at 37°C, before inversion into six-well plates containing tissue culture medium supplemented with 10% fetal calf serum for a further 10 hours. Subsequently cells were transfected with the TGF-β1 promoter-luciferase construct and 0.3 × 106 U937 cells were added directly into the insert thus accessing the basolateral aspect of the HK-2 cells in the absence (basal monos, solid bar) or presence of 10 μg/ml of anti-CD18 antibody (basal monos + CD18, cross-hatched bar). In control experiments serum-free medium alone was added to the transfected HK-2 cell monolayer (control, stippled bars). After a further 24-hour incubation, luciferase activity was quantified and TGF-β1 promoter activity was expressed as the relative change compared to the serum-free control. Results represent mean ± SD, n = 6.

Article Snippet: Other reagents and sources were as follows: testicular hyaluronidase (H3884) (Sigma, Poole, UK), recombinant human soluble ICAM-1 (R&D Systems Europe Ltd.), anti-human CD44 blocking monoclonal antibody BU75 (Ancell Corp., USA), anti-human CD18 blocking monoclonal antibody (Ancell Corp.), anti-human anti-CD54 (ICAM-1) cross-linking monoclonal antibody 6.5 (clone RR 6.5; a gift from Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, NJ), and anti-mouse IgG (whole molecule) (Sigma).

Techniques: Inhibition, Activity Assay, Blocking Assay, Transfection, Luciferase, Construct, Binding Assay, Labeling, Concentration Assay, Quantitation Assay, Radioactivity, Enzyme-linked Immunosorbent Assay, Incubation

Journal:

Article Title: Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-?1 Generation and Monocyte Binding

doi:

Figure Lengend Snippet: Removal of cell-surface HA increases ICAM-1-dependent TGF-β1 promoter activity. HK-2 cells transfected with the TGF-β1 promoter-luciferase construct were treated with bovine testicular hyaluronidase (final concentration, 200 μg/ml) at 37°C for 5 minutes before addition of U937 cells either alone (U937 + Hyal) or in the presence of either 400 ng/ml of soluble ICAM-1 (U937 + Hyal + sICAM-1), 5 μg/ml of irrelevant IgG1 antibody (U937 + Hyal + IgG), or 10 μg/ml blocking antibody to CD18 (U939 + Hyal + CD18Ab). In control experiments U937 cells were added to transfected HK-2 cells that had not received hyaluronidase treatment (control U937). TGF-β1 promoter activity was expressed as the relative change compared to the value obtained when U937 cells were added to the untreated HK-2 cells. Results represent mean ± SD, n = 6.

Article Snippet: Other reagents and sources were as follows: testicular hyaluronidase (H3884) (Sigma, Poole, UK), recombinant human soluble ICAM-1 (R&D Systems Europe Ltd.), anti-human CD44 blocking monoclonal antibody BU75 (Ancell Corp., USA), anti-human CD18 blocking monoclonal antibody (Ancell Corp.), anti-human anti-CD54 (ICAM-1) cross-linking monoclonal antibody 6.5 (clone RR 6.5; a gift from Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, NJ), and anti-mouse IgG (whole molecule) (Sigma).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Concentration Assay, Blocking Assay

Journal:

Article Title: Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-?1 Generation and Monocyte Binding

doi:

Figure Lengend Snippet: Removal of cell-surface HA increases ICAM-1-dependent monocyte binding. Confluent monolayers of serum-deprived HK-2 cells were treated with bovine testicular hyaluronidase (final concentration, 200 μg/ml) at 37°C for 10 minutes before addition 1 × 106 51Ci chromium-labeled U937 cells. The role of CD18-ICAM-1 interactions in monocyte binding was determined by pretreatment of monocytes with 400 ng/ml of soluble ICAM-1 or 10 μg/ml of blocking antibody to CD18 at 37°C for 1hour before their addition to HK-2 cells in the presence of either blocking reagent. In control experiments U937 cells were added to untreated HK-2 cells. Quantitation of bound radioactivity was done as described in Materials and Methods. Data represent mean ± SD of six individual experiments.

Article Snippet: Other reagents and sources were as follows: testicular hyaluronidase (H3884) (Sigma, Poole, UK), recombinant human soluble ICAM-1 (R&D Systems Europe Ltd.), anti-human CD44 blocking monoclonal antibody BU75 (Ancell Corp., USA), anti-human CD18 blocking monoclonal antibody (Ancell Corp.), anti-human anti-CD54 (ICAM-1) cross-linking monoclonal antibody 6.5 (clone RR 6.5; a gift from Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, NJ), and anti-mouse IgG (whole molecule) (Sigma).

Techniques: Binding Assay, Concentration Assay, Labeling, Blocking Assay, Quantitation Assay, Radioactivity

Journal:

Article Title: Renal Proximal Tubular Epithelial Cell Transforming Growth Factor-?1 Generation and Monocyte Binding

doi:

Figure Lengend Snippet: Human peripheral monocytes behave in an identical manner to U937 cells, increasing TGF-β1 promoter activity in an ICAM-1-dependent manner. A: Human peripheral monocyte increase in TGF-β1 activity is dependent on cell-cell contact and CD18. HK-2 cells were transfected with the TGF-β1 promoter-luciferase construct and 0.3 × 106 isolated human peripheral monocytes either added directly onto the HK-2 cell monolayer (contact, cross-hatched bars) or added onto a tissue culture insert (1.0 μm pore size) to prevent direct cell-cell contact (insert, solid bars). In addition 0.3 × 106 human peripheral monocytes pretreated with 10 μg/ml of anti-CD18 antibody (CD18) or irrelevant IgG1 control (IgG) for 1 hour at 37°C were added to transfected HK-2 cells. In control experiments serum-free medium alone was added to the transfected HK-2 cell monolayer (control, stippled bars). After a further 24-hour incubation, luciferase activity was quantified and TGF-β1 promoter activity expressed as the relative change compared to the serum-free control. Data represent mean ± SD, n = 6. B: Removal of cell-surface HA increases human monocyte-dependent TGF-β1 promoter activity. HK-2 cells transfected with the TGF-β1 promoter-luciferase construct were treated with bovine testicular hyaluronidase (final concentration, 200 μg/ml) at 37°C for 10 minutes before addition of human peripheral monocytes (0.3 × 106) either alone or in the presence of 400 ng/ml of soluble ICAM-1 or 10 μg/ml of blocking antibody to CD18 (U939 + Hyal + CD18). In control experiments, U937 cells were added to transfected HK-2 cells that had not received hyaluronidase treatment. In parallel experiments, human peripheral monocytes were pretreated with 5 μg/ml of anti-CD44 antibody alone or anti-CD44 antibody together with either soluble 400 ng/ml ICAM-1 or 10 μg/ml anti-CD18 antibody for 1 hour at 37 °C, before their addition to transfected HK-2 cells (again in the presence of the relevant antibodies). After a further 24-hour incubation, luciferase activity was quantified and TGF-β1 promoter activity expressed as the relative change compared to the value obtained when U937 cells were added to the untreated HK-2 cells. Data represent mean ± SD; n = 6; *, P < 0.05.

Article Snippet: Other reagents and sources were as follows: testicular hyaluronidase (H3884) (Sigma, Poole, UK), recombinant human soluble ICAM-1 (R&D Systems Europe Ltd.), anti-human CD44 blocking monoclonal antibody BU75 (Ancell Corp., USA), anti-human CD18 blocking monoclonal antibody (Ancell Corp.), anti-human anti-CD54 (ICAM-1) cross-linking monoclonal antibody 6.5 (clone RR 6.5; a gift from Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, NJ), and anti-mouse IgG (whole molecule) (Sigma).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Isolation, Incubation, Concentration Assay, Blocking Assay