Journal: PLoS ONE

Article Title: Integrin α D β 2 (CD11d/CD18) Is Expressed by Human Circulating and Tissue Myeloid Leukocytes and Mediates Inflammatory Signaling

doi: 10.1371/journal.pone.0112770

Figure Lengend Snippet: A. Whole venous blood from healthy volunteers was fixed and expression of α D β 2 on leukocyte subtypes was examined using mAb 169A (anti-α D ) and FITC-conjugated mAbs against CD14, CD15, and CD3 as described in . The percent of each cell type positive for α D β 2 is indicated by the bars. The error bars indicate the mean and SD of determinations using samples from four subjects. B. Monocytes were separated from venous blood of healthy volunteers and further separated into CD16 + and CD16 − CD14 + subpopulations as described in . Surface expression of α D β 2 , α L β 2 , α M β 2 , and α X β 2 was examined by flow cytometry using FITC- or ALEXA-488-conjugated antibodies and isotype-matched IgG controls. Cells in each monocyte fraction were also stained for CD14. These data indicate means and standard deviations in results from 3 experiments using samples from different subjects.

Article Snippet: Monoclonal antibodies (mAb) with reactivity against α D were developed and characterized as described , , and were generously provided by investigators at ICOS corporation.

Techniques: Expressing, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Integrin α D β 2 (CD11d/CD18) Is Expressed by Human Circulating and Tissue Myeloid Leukocytes and Mediates Inflammatory Signaling

doi: 10.1371/journal.pone.0112770

Figure Lengend Snippet: A. Leukocytes in whole cardiac blood from a mouse infected with the rodent malarial parasite Plasmodium berghei ANKA were stained for α D β 2 (arrows). B, C. Monocytes were first separated from an unfractionated mononuclear cell suspension from the peripheral blood of a healthy human volunteer and then further separated into CD16 + and CD16 − subpopulations as described in . The CD16 − CD14 + (B) and CD16 + (C) monocyte preparations were then fixed, permeabilized, and stained for α D (green fluorescence) using anti-α D mAb 169A. Propidium iodide was used to identify nuclei. In additional experiments isotype-matched non-immune IgG was used as the first immunoglobulin in the staining procedure to control for mAb 169A . In both monocyte subsets, α D β 2 staining had a granular pattern and in some areas there were large clusters of the integrin that appeared to be on or near the surface (arrows). An additional experiment indicated that α D β 2 also clusters on human neutrophils .

Article Snippet: Monoclonal antibodies (mAb) with reactivity against α D were developed and characterized as described , , and were generously provided by investigators at ICOS corporation.

Techniques: Infection, Staining, Suspension, Fluorescence, Control

Journal: PLoS ONE

Article Title: Integrin α D β 2 (CD11d/CD18) Is Expressed by Human Circulating and Tissue Myeloid Leukocytes and Mediates Inflammatory Signaling

doi: 10.1371/journal.pone.0112770

Figure Lengend Snippet: A. Tissue samples from a human subject with normal lungs who underwent autopsy after fatal head injury were fixed and stained for α D with mAb 169A as outlined in and . Microscopic evaluation revealed α D β 2 + macrophages in alveolar spaces (black arrow). There are also scattered α D + cells in alveolar walls (white arrows), which may be interstitial macrophages and/or dendritic cells. This image is representative of findings from analysis of lung tissue from three subjects who died without lung disease or injury. B. Autopsy samples from a patient who died with acute respiratory distress syndrome , were fixed, stained for α D , and examined by light microscopy. Numerous α D β 2 + macrophages were detected in alveolar spaces and walls. In some fields α D β 2 + neutrophils were also present (not shown). Lung samples from 3 patients who died with acute lung injury or ARDS as defined by consensus criteria were examined. We found α D β 2 + leukocytes in the alveoli in each case.

Article Snippet: Monoclonal antibodies (mAb) with reactivity against α D were developed and characterized as described , , and were generously provided by investigators at ICOS corporation.

Techniques: Staining, Light Microscopy

Journal: PLoS ONE

Article Title: Integrin α D β 2 (CD11d/CD18) Is Expressed by Human Circulating and Tissue Myeloid Leukocytes and Mediates Inflammatory Signaling

doi: 10.1371/journal.pone.0112770

Figure Lengend Snippet: Unfractionated human monocytes were incubated in wells coated with immobilized mAb against α D , or in wells coated with HSA or IgG1 as control conditions, for various times. Supernatants were collected and analyzed for IL-8 by ELISA. In some experiments engagement of α D β 2 was compared to engagement of other leukocyte integrins using immobilized mAb against individual leukocyte integrin α subunits. A. Engagement of α D β 2 by immobilized anti-α D mAb induced time-dependent release of IL-8, whereas IL-8 secretion by monocytes incubated on HSA- or IgG1-coated control surfaces was much lower. In parallel, incubation of monocytes with LPS (100 µg/ml) in suspension induced release of IL-8 at 8 (1.1 ng/ml) and 18 hr (4.4 ng/ml) (not shown). Engagement of αMβ 2 also induced time-dependent release of IL-8. B. Release of IL-8 triggered by engagement of α D β 2 on monocytes was dependent on the concentration of anti-α D mAb used to coat the wells. This figure indicates the results from an 8 hr incubation of monocytes on the triggering and control surfaces. C. Engagement of integrin α D β 2 was a potent stimulus for IL-8 secretion at 8 hr when immobilized mAb against α D were compared to immobilized mAb against other leukocyte integrin α subunits. Two activating anti-α D monoclonal antibodies, 169B and 217I, were examined in this experiment. The data in Panels A-C are individual determinations in single experiments. Data from additional experiments done at the 8 hr time point using monocytes from multiple different donors are shown in and . D. Wells were coated with human albumin or recombinant ICAM-3, monocytes were incubated in these wells for 18 hr. alone, in the presence of a blocking anti-α D mAb (mAb 240I), or in the presence of a non-blocking anti-α D monoclonal antibody (mAb 169A). IL-8 was then measured in the supernatants. The figure indicates the results of incubations with monocytes from 8 different subjects studied in 5 separate experiments on different days. Results from each subject are identified by a different symbol. The horizontal bars in the columns of data points indicate the means of determinations for each condition. The data were analyzed with Tukey's multiple comparison test and the Neuman-Kuels multiple comparison test, with similar results in each case. The significance values from the Neuman-Kuels analysis are shown (** = p<0.001; * = p<0.01). There was no difference in release of the IL-8 when monocytes were incubated with ICAM-3 alone versus incubation with ICAM-3 in the presence of the non-blocking mAb (p>0.05).

Article Snippet: Monoclonal antibodies (mAb) with reactivity against α D were developed and characterized as described , , and were generously provided by investigators at ICOS corporation.

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Suspension, Concentration Assay, Bioprocessing, Recombinant, Blocking Assay, Comparison

Journal: PLoS ONE

Article Title: Integrin α D β 2 (CD11d/CD18) Is Expressed by Human Circulating and Tissue Myeloid Leukocytes and Mediates Inflammatory Signaling

doi: 10.1371/journal.pone.0112770

Figure Lengend Snippet: Integrin α D β 2 was engaged by immobilized activating mAb and supernatants for mediator analysis were collected as outlined in . In (A) the concentration of anti-α D mAb, mAb against other leukocyte integrin α subunits, or control proteins used to coat the wells was 10 µg/ml, and in (B) the concentrations were 20 µg/ml. A. Engagement of integrin α D β 2 (8 hr) triggered release of MCP-1 that was much greater than that induced by immobilized mAb against other leukocyte integrin α subunits or release from monocytes incubated on control surfaces. Two activating anti-α D mAb, 169B and 217I, were studied. This result is representative of the pattern seen in eight separate experiments using monocytes from different donors, as shown in detail in . B. Engagement of integrin α D β 2 induced time-dependent release of IL-1β by monocytes. Two activating anti-α D mAb were examined, as in (A). In five additional experiments using monocytes from different donors, IL-1β protein was induced in monocyte lysates and released into the supernatants when integrin α D β 2 was engaged for 8 hr., and was greater than that in samples from monocytes incubated on control surfaces .

Article Snippet: Monoclonal antibodies (mAb) with reactivity against α D were developed and characterized as described , , and were generously provided by investigators at ICOS corporation.

Techniques: Concentration Assay, Control, Incubation