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

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

IgM binds AnnV⁺ PI⁺ and permeabilised human Jurkat cells.

<p>Human Jurkat cells were treated with camptothecin for 16-hours to induce apoptosis. Jurkat cells were then exposed to PBS or human serum as a source of IgM. IgM binding was then assessed by flow cytometry. Annexin-V (AnnV) and PI staining, assessed by flow cytometry, was used to determine the level of non-apoptotic (AnnV^<b>-</b>PI^<b>-</b>), early (AnnV^<b>+</b>PI^<b>-</b>) or late apoptotic (AnnV^<b>+</b>PI^<b>+</b>) thymocytes. <b>A</b>) Representative histogram illustrating IgM binding to AnnV^<b>+</b> apoptotic Jurkat cells within human serum and PBS treatment groups. <b>B</b>) Apoptotic Jurkat cells were gated on the basis of AnnV positivity and the percentage of AnnV^<b>+</b>IgM^<b>+</b> Jurkat cells was assessed. Apoptotic Jurkat cells exposed to human serum exhibited significant IgM binding. (Student’s <i>t</i>-test; <i>P</i> < 0.0001; <i>n</i> = 4) <b>C</b>) Samples exposed to human plasma consisted of three distinct populations: AnnV^<b>-</b>IgM^<b>-</b>, AnnV^<b>+</b>IgM^<b>-</b> and AnnV^<b>+</b>IgM^<b>+</b>. Apoptotic Jurkat cells were largely AnnV^<b>+</b>PI^<b>+</b>. <b>D</b>) The three distinct populations present in samples exposed to human serum were further characterised by examining FSC/SSC. Although both AnnV^<b>+</b>IgM^<b>-</b> and AnnV^<b>+</b>IgM^<b>+</b> populations were AnnV^<b>+</b>PI^<b>+</b> they occupy distinct separate regions of FSC/SSC contour plots. <b>E</b>) Confocal microscopy revealed that late apoptotic Jurkat cells exhibited AnnV and IgM staining which appeared to be restricted to the membrane. <b>F</b>) Non-apoptotic Jurkat cells were permeabilised with saponin prior to incubation with human serum. Permeability was confirmed by PI staining and IgM binding was assessed by flow cytometry. Following permeabilisation non-apoptotic Jurkat cells were predominantly PI positive. Non-permeable non-apoptotic Jurkat cells remained consistently IgM negative, whilst permeable Jurkat cells were IgM positive. Representative histograms and contour plots (<i>n</i> = 3 per group). Images were taken using a Leica SP5 with an x63/1.4 objective. Whole image contrast/brightness was adjusted using ImageJ or Adobe Photoshop. Data expressed as means ±SEM.</p

IgM binds strongly to permeabilised non-apoptotic and apoptotic thymocytes.

<p>Non-apoptotic and apoptotic mouse thymocytes, induced by overnight culture, were permeabilised with saponin prior to incubation with Balb/c plasma. Permeability was confirmed by PI staining and IgM binding was assessed by flow cytometry. For confocal microscopy AnnV staining was used to indicate permeabilisation <b>A</b>) Following permeablisation non-apoptotic thymocytes were predominantly PI positive. Non-permeable non-apoptotic thymocytes remained consistently IgM negative, whilst the majority of permeable thymocytes were IgM positive. Inclusion of isotype control IgM antibodies (mouse (Balb/c) IgM κ isotype control) confirmed that IgM binding was not due to a non-specific interaction of IgM. <b>B</b>) A small proportion of apoptotic thymocytes were PI positive representing late apoptotic thymocytes; however following permeabilisation with saponin the cell population became PI positive. Whilst a minority of apoptotic thymocytes were IgM positive, representing the AnnV^<b>+</b>IgM^<b>+</b> population discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131849#pone.0131849.g001" target="_blank">Fig 1C–1E</a>, with permeabilisation the population became IgM positive. <b>C</b>) Confocal microscopy demonstrates that permeabilised thymocytes were AnnV positive, used to indicate permeability, with IgM appearing to bind part of the cellular membrane. Representative histograms (Non-apoptotic thymocytes <i>n</i> = 3; Apoptotic thymocytes <i>n</i> = 4). Images were taken using a Leica SP5 with an x63/1.4 objective. Whole image contrast/brightness was adjusted using ImageJ or Adobe Photoshop.</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

IgM binds to permeable human proximal tubule epithelial (HK-2) cells.

<p>Non-apoptotic HK-2 cells (<b>A</b>) were permeabilised with saponin prior to incubation with human serum (as a source of IgM). Permeability was confirmed by PI staining and IgM binding was assessed by flow cytometry. <b>B</b>) Saponin treated HK-2 cells demonstrated a distinct shift in PI positivity indicating successful permeabilisation. <b>C</b>) Non-permeable HK-2 cells remained consistently IgM negative, whilst HK-2 cells treated with saponin were IgM positive. Representative histograms and contour plots (<i>n</i> = 3 per group). Images were taken using a Zeiss Axiovert S100 with an x32/0.4 objective and CoolSnap RS Photometrics camera. Whole image contrast/brightness was adjusted using ImageJ.</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

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 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

IgM rapidly and specifically binds to a distinct population of AnnV⁺PI⁺ mouse thymocytes.

<p>Mouse thymocytes were rendered apoptotic by overnight culture. Thymocytes were then exposed to either mouse anti-human isotype control antibodies (mouse (Balb/c) IgM κ isotype control), PBS, Balb/c plasma (as a source of IgM) or plasma from immunodeficient RAG1-deficient mice. IgM binding was then assessed by flow cytometry. Annexin-V (AnnV) and propidium iodide (PI) staining, assessed by flow cytometry, was used to determine the level of non-apoptotic (AnnV^<b>-</b>PI^<b>-</b>), early (AnnV^<b>+</b>PI^<b>-</b>) or late apoptotic (AnnV^<b>+</b>PI^<b>+</b>) thymocytes. <b>A</b>) Representative histograms illustrating IgM binding to AnnV^<b>+</b> apoptotic thymocytes within treatment groups. <b>B</b>) Apoptotic thymocytes were gated on the basis of AnnV positivity and the percentage of AnnV^<b>+</b>IgM^<b>+</b> thymocytes was assessed. IgM binding in isotype, RAG1-deficient mice plasma and PBS treatment groups remained non-significant whilst samples exposed to Balb/c plasma exhibited significant binding (Kruskal–Wallis one-way ANOVA; <i>P</i> < 0.0001). However, surprisingly approximately 68% were IgM^<b>-</b>. <b>C</b>) Samples exposed to Balb/c plasma consisted of three distinct populations: AnnV^<b>-</b>IgM^<b>-</b>, AnnV^<b>+</b>IgM^<b>-</b> and AnnV^<b>+</b>IgM^<b>+</b>. <b>D</b>) The three distinct populations present in samples exposed to Balb/c plasma were further characterised by examining the FSC/SSC profile and AnnV and PI positivity. Although both AnnV^<b>+</b>IgM^<b>-</b> and AnnV^<b>+</b>IgM^<b>+</b> populations were predominantly AnnV^<b>+</b> PI^<b>+</b> they occupied distinct separate regions of FSC/SSC contour plots. <b>E</b>) Within the AnnV^<b>+</b>IgM^<b>+</b> population IgM binding occurred rapidly (<i>n</i> = 4). Data representative of <i>n</i> = 5 isotype, <i>n</i> = 6 RAG1 deficient mice plasma + Anti-IgM, <i>n</i> = 8 PBS + Anti-IgM and <i>n</i> = 8 Balb/c plasma + Anti-IgM. Data expressed as means ±SEM.</p