The Aminopeptidase CD13 Induces Homotypic Aggregation in Neutrophils and Impairs Collagen Invasion

<div><p>Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation <i>in vivo</i>.</p></div

Anti-CD13 mAbs induce neutrophil HA.

<p>Neutrophils were incubated with bestatin (0.1 mM), WM-15 or IgG1 (8 μg/ml), WM-47 (1:40 v/v), MY7 (5 μg/ml) or vehicle control. HA was assessed by light microscopy in duplicate at 37°C and 4°C (A) and flow cytometry (E and F) and data represent the mean ± SEM of <i>n</i> = 3 independent experiments. ***, <i>P</i> < 0.001, **, <i>P</i> < 0.01 vs. control, by two-way ANOVA/Bonferroni test (A). Representative photomicrographs after 60 min at 37°C (B and C). Original magnification x 50. Representative SSC area by FSC area dot plots of the fixed neutrophil population separated into singlets and non-singlets (D). Quantification of neutrophil HA at 30 min. ***, <i>P</i> < 0.001, **, <i>P</i> < 0.01 vs. control, by repeated measures ANOVA/Dunnett’s test (E and F).</p

Effect of kinase inhibitors on CD13-mediated neutrophil HA.

<p>Neutrophils were pre-incubated for 15 min with LY294002 (10 μM), UO126 (10 μM), or vehicle control prior to the addition of WM-15 (8 μg/ml), WM-47 (1:40 v/v), MY7 (5 μg/ml) or buffer. HA was assessed at 30 min by flow cytometry. Data represent the mean ± SEM of <i>n</i> = 4 (A) and <i>n</i> = 6 (B) independent experiments. *, <i>P</i> < 0.05, ns, non-significant, by Student’s paired <i>t</i> test.</p

Effect of CD13 ligation on neutrophil and recombinant human CD13 activity.

<p>Neutrophils were pre-incubated with bestatin (0.1 mM), anti-CD13 mAb WM-15 (8 μg/ml), anti-CD13 mAb MY7 (5 μg/ml), anti-CD13 mAb WM-47 (1:40 v/v) or vehicle control for 30 min. Cleavage of L-Alanine 4-nitroanilide (2 mM) was measured over 60 min and is expressed as a percentage of the activity observed with control treated cells. Data represent the mean ± SEM of <i>n</i> = 7 (Bestatin), <i>n</i> = 3 (WM-15 and WM-47) or mean ± SD of <i>n</i> = 2 independent experiments (MY7) each performed in triplicate (A). rCD13 (200 ng/ml) was pre-incubated alone or with increasing concentrations of bestatin for 30 min. Cleavage of L-Alanine 4-nitroanilide was measured over 60 min and converted to nmol substrate/min. The mean ± SEM from three independent experiments, each performed in triplicate, are shown. (B). ***, <i>P</i> < 0.001, **, <i>P</i> < 0.01, ns, non-significant vs. control, by Student’s paired <i>t</i> test.</p

CD13-mediated neutrophil HA depends on CD13 cross-linking, involves actin polymerisation and CD13 directly participates in the adhesive interaction.

<p>Neutrophils were pre-incubated for 30 min with cytochalasin-D (2 μM) or vehicle control prior to the addition of WM-15 (8 or 100 μg/ml), Fab fragments of WM-15 (8 μg/ml) or buffer (A). Neutrophils were incubated with WM-15 (8 μg/ml) or with WM-47 alone or followed by a 30 min incubation with F(ab)’₂ fragments of GAM antibody (B). HA was assessed at 30 min by flow cytometry. Data represent the mean ± SEM of at least <i>n</i> = 3 independent experiments (GAM <i>n</i> = 2). ***, <i>P</i> < 0.001, **, <i>P</i> < 0.01, *, <i>P</i> < 0.05 vs. WM-15, by Student’s unpaired <i>t</i> test (A). ns, non-significant, by Student’s unpaired <i>t</i> test (B). Neutrophils were pre-incubated with WM-15 (8 μg/ml) or vehicle control for 10 min and added to PBS- or rCD13 (1000 ng/ml) coated wells in duplicate for 20 min. The number of adherent neutrophils was assessed by light microscopy at 100x magnification. *, <i>P</i> < 0.05 vs. control, by repeated measures ANOVA/Dunnett’s test (C).</p

Effect of CD13 ligation on neutrophil shape change and migration.

<p>Neutrophils were pre-incubated with bestatin (0.1 mM), WM-15 or IgG1 (8 μg/ml), WM-47 (1:40 v/v), MY7 (5 μg/ml) or vehicle control for 30 min (A, B, C and D) or 10 min (E and F). Cells were stimulated in triplicate with a concentration range of fMLP or IL-8 for 10 min and percentage shape change assessed by flow cytometry (A and B). Chemotaxis was assessed in duplicate using a ChemoTx^® plate and migration towards a concentration range of IL-8 quantified after 90 min by manual counting (C and D). Migration through collagen I, gelled in a Transwell^® insert, towards IL-8 (100 ng/ml) was quantified in duplicate after 9 hr and is expressed as a percentage of the migration observed with control treated cells (E and F). Data represent the mean ± SEM of <i>n</i> = 3 independent experiments (A, C, D, E and F) or mean ± SD of <i>n</i> = 2 independent experiments (B). ***, <i>P</i> < 0.001, *, <i>P</i> < 0.05, ns, non-significant vs. control, by repeated measures ANOVA/Tukey’s test.</p

Cognitive triggers of auditory hallucinations: An experimental investigation

It has proved difficult to establish the internal process by which mental events are transformed into auditory hallucinations. The earlier stages of the generation of hallucinations may prove more accessible to research. Cognitions have been reported by patients as a trigger of auditory hallucinations, but the role of these preceding thoughts has not been causally determined. Therefore, the role of cognition in triggering auditory hallucinations was tested in an experimental study. Thirty individuals who experienced auditory hallucinations in social situations entered a neutral social situation presented using virtual reality. Participants randomised to the experimental condition were instructed to think their hallucination-preceding thoughts, and those randomised to the control condition were instructed to think neutral thoughts. Twenty-seven participants (93%) were able to spontaneously identify a cognition which preceded a hallucination. There was no difference between the experimental and control groups in the occurrence or severity of auditory hallucinations in virtual reality. Virtual reality did not lead to physical side effects or an increase in anxiety. The relationship between antecedent cognitions and auditory hallucinations is likely to be more complex than the one tested. It is argued that the effect of cognition on auditory hallucinations may be mediated by affect but this needs to be investigated through further experimental research

Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis - Fig 5

<p><b>A.</b> iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad3 to the <i>ITGB6</i> promoter at approximately -936 to -755 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments. <b>B</b>. iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad3 to the <i>ITGB6</i> promoter at approximately -1608 to -1500 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments. <b>C.</b> iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad4 to the <i>ITGB6</i> promoter at approximately -936 to -755 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments. <b>D.</b> iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad4 to the <i>ITGB6</i> promoter at approximately -1608 to -1500 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments. <b>E.</b> iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad2 to the <i>ITGB6</i> promoter at approximately -936 to -755 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments. <b>F.</b> iHBECs were treated with 2 ng/ml TGFβ1 and binding of Smad2 to the <i>ITGB6</i> promoter at approximately -1608 to -1500 was assessed after 0, 0.5, 1 and 2 hours using ChIP. Data are expressed as mean relative binding (relative to 0 h) ± SEM from 3 independent experiments.</p