12 research outputs found
Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells
A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1–2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes
The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells:CD14 and rapid tethering of apoptotic cells
Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. © 2013 Thomas et al
188 The inflammatory response of in vitro co-culture models of normal and cystic fibrosis (CF) airways to CF-relevant pathogens
CD14 is expressed in non-myeloid cells but is not sufficient for inducible LPS responses.
<p>(A) Flow cytometric analysis of cell surface CD14 expression on a panel of non-myeloid cells. Cells were assessed for cell surface CD14 through the use indirect immunofluorescence with anti-CD14 mAb 63D3 (or MOPC21 isotype control) and detected with goat anti-mouse-phycoerythrin. Frequency histograms of at least 5000 events are shown for each cell type (red: CD14; grey: IgG1/κ isotype control). Numerical values shown are the mean fluorescence intensity for anti-CD14 (open black) or isotype control (solid grey) stained cells. (B) IL-8 production by CD14-expressing BEAS-2B and Calu-3 cells following LPS treatment at the indicated concentrations for 24 hours. ELISA of supernatant IL-8 was undertaken and results shown are mean ± SE of three independent experiments. (C) Flow cytometric analysis of cell surface TLR4 expression on BEAS-2B and Calu-3 cells. Cells were assessed for cell surface TLR4 by direct immunofluorescence with PE-conjugated anti-TLR4 mAb HTA125 (or an isotype control). Frequency histograms of at least 5000 events are shown for each cell type (open black: TLR4-PE; solid grey: IgG2a/κ isotype control-PE). Values shown are the mean fluorescence intensity for anti-TLR4 (black) or isotype control (grey) stained cells.</p
All THP-1 cell-derived macrophages utilise CD14 for interaction with apoptotic cells.
<p>UV-induced apoptotic BL cells (>80% apoptotic as assessed by nuclear morphology) were co-cultured with THP-1 phagocytes in the presence of the indicated mAbs (anti-CD14 mAb 61D3, a blocking CD14 mAb, or anti-CD14 mAb 63D3, a non-blocking, isotype matched control). Following co-culture (1h) at 37°C unbound apoptotic cells were removed and the interaction of phagocytes with apoptotic cells assessed by light microscopy of Jenner-Giemsa stained cells. (A) shows the percentage of phagocytes interacting with apoptotic cells. (B) shows the effect of 61D3 and 63D3 on apoptotic cell interaction with phagocytes (% of AC alone). (C) Compares the 61D3 inhibition as a measure of CD14 function for each macrophage type. All data shown are mean ± SE for 7 independent experiments. Statistical analyses used ANOVA with Bonferroni post test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p
Characterisation of THP-1 macrophage phenotype and LPS responses.
<p>THP-1 monocytes (THP-1) cells were stimulated to differentiate in the presence of dihydroxyvitamin D3 (VD3), phorbol ester (PMA) or both (VD3/PMA) for 72 hours prior to analyses. (A) Flow cytometric analysis of cell surface CD14 expression using indirect immunofluorescence with mAb 63D3 detected with anti-mouse-phycoerythrin. Frequency histograms of at least 5000 events are shown for each cell type (open black: CD14; solid grey: IgG1/κ isotype control). Data shown are representative of at least three independent experiments. (B) Production of TNF-α in response to a range of LPS concentrations for 4h in the presence of 10% v/v normal human serum, detected by ELISA (data shown are mean ± SE for 3 independent experiments). (C) Flow cytometric analysis of cell surface TLR-4 using direct immunofluorescence with mAb HTA-125-phycoerythrin (open black: anti-TLR-4; solid grey: IgG2a/κ isotype control). Data shown are representative of at least three independent experiments.</p
LPS but not apoptotic cells activates NFκB inflammatory signalling.
<p>HeLa cells were transfected with both the luciferase NFκB reporter plasmid and a CD14WT expression plasmid or ICAM-3 expression plasmid as a control using <i>Trans</i>IT LT-1. Expression was allowed to proceed for 24 hours prior to further analyses. (A) CD14 expression was assessed using indirect immunofluorescence with mAb 63D3 (open black) detected using goat anti-mouse PE, compared to isotype control stained cells (solid grey). (B) Cells were treated with either 100µg/ml of LPS or apoptotic human B cells for 5h prior to assessing NFκB-mediated transcriptional activity with One-Glo Luciferase assay system. Apoptotic cells were in excess of 80% apoptotic by nuclear morphology. Relative light units were quantified using a microplate luminometer. The data shown is mean ± SE of three independent experiments. Statistical analyses used ANOVA with Tukey post-test. **<i>P</i><0.001; ***<i>P</i><0.001; ns = not significant.</p
Residue 11 of CD14 is essential for binding to apoptotic cells.
<p>HeLa cells were used as surrogate phagocytes and co-cultured with apoptotic human B cells. Following co-culture at 4°C unbound apoptotic cells were removed and the interaction of phagocytes with apoptotic cells assessed by light microscopy of Jenner-Giemsa stained cells. (A) HeLa cells (mock or CD14WT transfected) co-cultured with UV-induced apoptotic human B cells (>80% apoptotic as assessed by nuclear morphology) for 5 or 15 min were used to assess the optimal period to reveal the role of CD14 to promote apoptotic cell binding. Data shown are mean ± SE for three independent experiments. Statistical analyses used ANOVA with Tukey post-test. **<i>P</i><0.01. (B) HeLa cells transfected with CD14WT or point mutants were used as surrogate phagocytes and co-cultured with apoptotic human B cells for 5 min. Following co-culture at 4°C unbound apoptotic cells were removed and the interaction of phagocytes with apoptotic cells assessed by light microscopy of Jenner-Giemsa stained cells. Data are shown as the percentage of HeLa cells interacting with apoptotic cells (mean ± SE above the binding to HeLa mock) for three independent experiments. Statistical analyses used ANOVA with Dunnett’s post-test. *<i>P</i><0.05; **<i>P</i><0.01.</p
Mapping of key residues within CD14 that are required for binding of mAbs 61D3 and MEM18.
<p>Monoclonal Abs 61D3 and MEM18 were tested for reactivity against wild-type CD14 and a panel of point mutants. (A) Anti-human Fc immobilised soluble CD14-Fc fusion proteins were probed by ELISA with 61D3 or MEM18 and mAb binding detected with anti-mouse-HRP prior to developing with OPD substrate and reading OD492nm. mAb is shown as a ratio of 63D3 binding to control for protein expression. Data shown are mean ± SE of three independent experiments and mAb (61D3 or MEM18) binding (relative to 63D3) is expressed as a % of binding to wtCD14Fc. Statistical analysis conducted was ANOVA followed by Dunnett’s post-test (**<i>P</i><0.01 compared to wtCD14). (B) HeLa cells were transfected with the indicated membrane CD14 (WT or mutant) prior to staining with IgG1/κ isotype control MOPC21, anti-CD14 mAb 63D3, or (C) anti-CD14 mAb MEM18. In each case, mAb binding was detected using goat anti-mouse-PE and flow cytometry. Fluorescence data are shown as histograms with at least 5000 events per plot and are representative of at least three independent experiments.</p
Extramyeloid CD14 is functional to mediate interaction with apoptotic cells.
<p>Non-myeloid cells (epithelial cells: BEAS-2B, Calu-3, H400, MCF-7 HeLa or human pulmonary fibroblasts, HPF) were seeded to glass slides and co-cultured with apoptotic human B cells in the absence or presence of anti-CD14 mAbs 61D3 or 63D3. Following co-culture for 1h at 37°C, unbound apoptotic cells were removed by washing and the percentage of non-myeloid phagocytes interacting with apoptotic cells was assessed by light microscopy of Jenner-Giemsa stained cells. Results shown are mean ± SE of three independent experiments. Statistical analyses used ANOVA with Tukey post-test. *<i>P</i><0.05; **<i>P</i><0.01.</p