Thioglycollate-induced cell recruitment patterns in CD44 −/− animals.

<p>Peritonitis was induced in wild type (C57BL/6J) or CD44−/− animals by injection of 2.5 ml of thioglycollate. At time points indicated, estimates of the total cell counts and percentage of cell types present in the peritoneal lavage fluid was made by microscopy and flow cytometry and the total counts for different cell populations calculated. Data are mean cell numbers ± SEM from 5 separate experimental animals. Black bars: neutrophils, dark grey bars: macrophages, grey hatched bars: lymphocytes, white hatched bars: eosinophils, other bar: mast cells.</p

CD44 variant antibodies do not promote phagocytosis of apoptotic PMN.

<p>A) Monocytes (white bars) or monocyte-derived macrophages cultured for 5 days in the absence (black bars) or presence of dexamethasone (gray bars) were incubated with different isoform-specific CD44 mAb or CD64 mAb (10.1) for 30 min to allow saturation of binding prior to addition of FITC-conjugated F(ab′)₂ goat anti-mouse immunoglobulins and assessment of binding by flow cytometry. Data shown are the average mean fluorescence intensity ± SEM for 4 independent experiments. B) Monocyte-derived macrophages were incubated with isoform-specific CD44 mAb for 30 min to allow saturation of binding prior to addition CMFDA-labelled apoptotic targets at 37°C for the times indicated. Assessment of phagocytosis was made by flow cytometry. Data shown are mean percent phagocytosis ± SEM from 4 different macrophage preparations, no significant augmentation of phagocytosis compared with control was observed with any of the variant antibodies examined (NS).</p

CD44 promotes a serum-opsonin independent mechanism of phagocytosis of apoptotic neutrophils.

<p>A) Monocyte-derived macrophages were incubated in the absence or presence of CD44 mAb for 30 min prior to addition CMFDA-labelled neutrophils that had been cultured either in human serum albumin (Albumin) or autologous serum (Serum). Assessment of phagocytosis was made by flow cytometry, data are mean ± SEM from 5 independent experiments. Although CD44 augmented phagocytosis relative to untreated cells (* = p<0.05), no significant differences between phagocytosis of PMN cultured in albumin versus serum was found following CD44 treatment. B) Glucocorticoid-treated monocyte-derived macrophages were incubated in the absence or presence of CD44 mAb for 30 min prior to addition CMFDA-labelled neutrophils that had been cultured in human serum albumin. Phagocytosis was assessed in the presence of 100 ng/ml of protein S for 30 min and the proportion of phagocytic macrophages determined by flow cytometry. Data shown are mean percentage phagocytosis ± SEM from 5 independent experiments. Although the presence of protein S augmented phagocytosis by glucocorticoid-treated macrophages (* = p<0.05), there was no significant difference between phagocytosis of PMN in the presence or absence of protein S following CD44 treatment.</p

Effect of CD44 on macrophage migration.

<p>A) Monocyte-derived macrophages were incubated in the absence or presence of CD44 mAb for 30 min to binding prior to “wounding” of the cell monolayer. Assessment of migration into the wounded area was made by microscopy at 18 h, data shown are mean number of cells present in the wound ± SEM from 6 separate experiments. In paired analysis there is a significant reduction of migration in the presence of CD44 mAb (p<0.05). B) Monocyte-derived macrophages were incubated in the absence or presence of CD44 mAb for 30 min to binding prior to “wounding” of the cell monolayer. Assessment of macrophage migration into the wounded area was made by quantification of the numbers of macrophages in the wound using time lapse microscopy images captured over 10 hours. Measurements for untreated macrophages are indicated by gray diamond symbols and CD44-treated macrophages by black squares. Data are mean number of cells observed in the wound ± SEM from 3 independent experiments. * indicates that migration was significantly reduced by CD44 mAb from 3 h onwards (p<0.05). Monocyte-derived macrophages adherent to glass coverslips were treated with media (C) or CD44 mAb (D) for 30 min prior to fixation with paraformaldehyde and staining for filamentous actin with rhodamine phalloidin. Representative micrographs show localisation of podosome-like structures in adherent macrophages (C) and marked redistribution that is observed following CD44 antibody binding (D). Scale bar = 10 µm.</p

Effects of CD44 cross-linking on phagocytosis of human apoptotic PMN transfered into the peritoneal cavity of mice.

<p>A) CMFDA-labelled human apoptotic neutrophils were transferred into the peritoneal cavity of mice that had been previously injected with CD44 mAb, 8D2 or mouse IgG1 control antibody. Phagocytosis of apoptotic cells was determined by flow cytometry following labelling of mouse macrophages with PE-conjugated F4/80. Representative histograms show Forward Scatter versus FL-1 for IgG1 treated versus 8D2 treated animals – representative of 4 independent experiments that were performed. B) Wild-type (C57BL/6J) or CD44−/− mice were pre-injected with either IgG1 (white bars) or CD44 mAb (8D2 – black bars) into the peritoneal cavity prior to injection of human apoptotic neutrophils. Quantification of phagocytosis by peritoneal macrophages lavaged from either wild type (C57BL/6J) or CD44 −/− was made by flow cytometry as described for (A) above. For C57BL/6J (A) results shown are the mean ± SEM for 10 separate animals. For C57BL/6J (B) and CD44 −/−, results are from 4 independent experiments. ** indicates results are statistically significant (p<0.01).</p

CD44 cross-linking promotes human macrophage phagocytosis of apoptotic human PMN.

<p>A) Monocyte-derived macrophages were incubated with CD44 mAb 5A4 Fab′ fragments (squares) or control Fab′ (triangles) for 30 min prior to addition of F(ab′)₂ goat anti-mouse immunoglobulins (black symbols, solid line) or PBS control (gray symbols, dashed lines). Macrophages were then co-incubated with apoptotic targets at 37°C for the times indicated prior to assessment of phagocytosis by microscopy. Results shown are mean ± SEM for n = 5 separate experiments. ** indicates significant difference between cross-linking versus non-cross-linked (p<0.01). B) Monocyte-derived macrophages were incubated with CD44 mAb for 30 min to allow saturation of binding prior to addition of CMFDA-labelled apoptotic targets at 37°C for the times indicated. Assessment of phagocytosis was made by flow cytometry (white bars = untreated; gray bars (CD44-treated). Data shown are mean percentage phagocytosis ± SEM for 3 independent experiments. At all time points examined except 5 min, CD44-treated cells exhibit significant augmentation of phagocytosis (p<0.05).</p

Effects of pharmacological inhibition of signalling pathways in macrophages upon CD44-augmentation of phagocytosis.

<p>A) Monocyte-derived macrophages were incubated with CD44 mAb for 30 min prior to addition of dibutyryl cAMP at concentrations shown. Macrophages were then co-incubated with apoptotic targets at 37°C for 30 min prior to assessment of phagocytosis by microscopy. Results shown are mean percentage phagocytosis for 3 independent experiments. B) Monocyte-derived macrophages were pre-incubated with 10 µM LY294002 at 37°C and then co-incubated with CD44 mAb for 30 min prior to addition of CMFDA-labelled apoptotic targets. After 30 min, assessment of phagocytosis was made by flow cytometry. Results shown are mean percentage phagocytosis ± SEM for 5 independent experiments. C)Monocyte-derived macrophages were pre-incubated with 50 nM PD98059 at 37°C and then co-incubated with CD44 mAb for 30 min prior to addition of CMFDA-labelled apoptotic targets. After 30 min, assessment of phagocytosis was made by flow cytometry. Results shown are mean percentage phagocytosis ± SEM for 3 independent experiments. D) Monocyte-derived macrophages were pre-incubated with 25 µM PP2 at 37°C and then co-incubated with CD44 mAb for 30 min prior to addition of CMFDA-labelled apoptotic targets. After 30 min, assessment of phagocytosis was made by flow cytometry. Results shown are mean percentage phagocytosis ± SEM for 4 independent experiments. E) Monocyte-derived macrophages were pre-incubated with 25 µM Genistein at 37°C and then co-incubated with CD44 mAb for 30 min prior to addition of CMFDA-labelled apoptotic targets. After 30 min, assessment of phagocytosis was made by flow cytometry. Results shown are mean percentage phagocytosis ± SEM for 3 independent experiments. In panels B-E there was no statistical difference between the percentage phagocytosis recorded in the presence or absence of pharmacological inhibitor following CD44 mAb treatment.</p

Augmentation of phagocytosis by CD44 in Tiam-1 −/− mice.

<p>A) Peritoneal macrophages from wild-type (FVB) and Tiam-1 −/− mice were pre-treated with CD44 mAb IM7.8.1 or rat IgG2b control prior to incubation with apoptotic human PMN. After 30 min phagocytosis of apoptotic PMN was determined by microscopy. Results show mean percentage phagocytosis ± SEM for macrophages derived from 4 separate animals. B) Representative microscopy images showing Tiam1 −/− peritoneal macrophages that have been pre-treated with or without CD44 mAb IM7.8.1 prior to incubation with apoptotic human PMN and then stained for myeloperoxidase activity. Scale bar = 20 µm.</p

CD44-augmentation of phagocytosis is associated with phosphorylation of paxillin and Rac2 activation.

<p>A) Monocyte-derived macrophages were incubated with CD44 mAb at 37°C for different time points up to 60 min prior to lysis in RIPA buffer. Immunoblot analysis of tyrosine phosphorylation of paxillin revealed a time-dependent increase in phosphorylation following CD44 antibody binding. Image shows representative autoradiograph of a single experiment from 3 that were performed. Densitometric analysis of the phosphopaxillin using ImageJ (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) confirms increased phosphorylation of paxillin following CD44 treatment. For control phosphopaxillin levels at 0, 15, 30, 45 and 60 min was 7.7, 9.9, 9.3 and 8.2 respectively. In contrast, phosphopaxillin levels following CD44 cross-linking were 3.7, 8.8, 13.7, 12.4 and 15.6 at 0, 15, 30, 45 and 60 min respectively. Molecular weight standards shown in KDa. B) Monocyte-derived macrophages were incubated with either control IgG1 or CD44 mAb at 37°C for different times up to 20 min prior to lysis in RIPA buffer as indicated. Pull down assays using PAK CRIB agarose beads revealed a robust increase in GTP-bound Rac2 in the presence but not absence of CD44 antibody binding. A representative autoradiograph from a single experiment (from 5 that were performed) is shown.</p

Xanthohumol Induces Apoptosis in Human Malignant Glioblastoma Cells by Increasing Reactive Oxygen Species and Activating MAPK Pathways

The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-l-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme