Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Expression profile of shed MVs in multiple trauma. (A) Representative dot plot of a healthy control (left) and a multiple trauma patient (right). Annexin V- positive MV fraction was analyzed for CD66b and C5aR1 expression. FITC was used for C5aR1 staining, and APC-Cy7 for CD66b. Q2 represents AnnexinV + , C5aR1 + , CD66b + MVs positive MV fraction. (B) Quantitative analysis for granulocyte-derived MVs and (C,D) granulocyte-derived MVs expressing C5aR1 in 5 μl of MV enriched plasma, respectively. n = 8–10, * p < 0.05 compared to healthy.

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Expressing, Control, Staining, Derivative Assay, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: C5a alters MV shedding in PMNs. (A) PMNs were stimulated for 0, 10, 30, 60, and 240 min with C5a (100 ng/ml) or LPS (5 μg/ml) and supernatants were analyzed by FACS. Untreated cells served as control. n = 10, One-Way ANOVA followed by Student-Neuman-Keuls post-hoc testing was performed for each time point, * p < 0.05. (B) MVs from supernatants were further analyzed for expression of C5aR1 by FACS. n = 3 per group, * p < 0.05. (C) Heat map and gene list of selected and differentially regulated genes upon C5a treatment in in neutrophils after 1 h ( n = 3 per group).

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Control, Expressing

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Effect of the C5aR1-antagonist PMX53 on vesicle shedding. (A) Upper panel shows merged image of F-actin staining (red, Phalloidin-Alexa Flour 568) and nuclei staining (DAPI) of fixed PMNs, untreated, after treatment with native human C5a (100 ng/ml), and C5a treatment in combination with PMX53 (10 μM) for 1 h, respectively at 630x magnification. Lower panel with further zoom-in on PMNs from the upper panel, white arrows indicate shed MVs. Experiment was repeated at least three times. (B) Quantitative analysis of shed MV at different C5a concentrations and in presence of the PMX53 together and alone in 10 μl supernatant ( n = 3). * p < 0.05.

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: PMX53 and NAV2729 show similar effects in blocking C5aR1-positive MV shedding. Representative flow cytometry panels for C5aR1 expression (FITC) (A) on PMNs or (B) in PMN supernatants including C5aR1 + MV counts in Q2 after 1 h stimulation in the presence of PMX53. Quantitative analysis of flow cytometry data showing (C) C5aR1 expression on PMN surfaces (FITC MFI) and (D) C5aR1 + MV counts. Quantitative flow cytometry analysis including the MV-specific inhibitor NAV2729 (10 μM) (E) for C5aR1 expression (FITC MFI) on PMNs and (F) C5aR1 + MV counts in 10 μl PMN supernatants after 1 h, n = 4 per group, respectively. * p < 0.05.

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Blocking Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: C5aR1 expression and MV shedding after incubation of PMNs with serum obtained from PT patients (S PT) and healthy donors (S Healthy). PMNs were incubated in HBSS++ buffer supplemented with 20% of pooled human serum from PT patients or from healthy donors for 1 h. (A) Quantification of C5aR1 expression on PMNs and (B) C5aR1 + MV counts in 10 μl of PMN supernatants after incubation. n = 4, * p < 0.05.

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Complement C5a Induces Pro-inflammatory Microvesicle Shedding in Severely Injured Patients

doi: 10.3389/fimmu.2020.01789

Figure Lengend Snippet: Model for C5a-mediated alterations on MV shedding for neutrophils after PT. PT causes an early activation of complement generating increased C5a concentrations. C5a induces activity of the small GTPase Arf6, which is dependent on C5aR1 signaling. Additionally, specific changes in gene expression pattern are involved in MV shedding. Resting PMNs show physiological MV shedding and composition, which have no inflammatory potential. In contrast, PMNs incubated with C5a loose C5aR1 surface expression induced by C5aR1 signaling and Arf6-mediated shedding of C5aR1-positive MVs. Furthermore, these MVs show increased CD66b and C5aR1 expression, which show pro-inflammatory features when incubated with resting PMNs. C5a induces shedding of MVs, which lead to NAPDH oxidase activation, ROS generation and MPO release in resting PMNs. Moreover, IL-6 generation was observed when these MVs were incubated in whole blood. PMX53 and the Arf6-selective compound NAV2729 can block the shedding of C5aR1-positive MVs from PMN surfaces and its inflammatory effects.

Article Snippet: MVs were stained with mouse anti-human C5aR1-FITC antibody (MCA2059F, Bio-Rad) and mouse anti-human CD66b-APC-Cy7 (305126, Biolegend) or the respective isotype controls in AV binding buffer.

Techniques: Activation Assay, Activity Assay, Gene Expression, Incubation, Expressing, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Complement Regulation in Human Tenocytes under the Influence of Anaphylatoxin C5a

doi: 10.3390/ijms22063105

Figure Lengend Snippet: Antibodies and dyes used.

Article Snippet: Mouse anti-human C5aR1 (CD88) , GeneTex, Biozol, Eching, Germany , GTX74845 , 1 mg/mL , 1:20.

Techniques: Concentration Assay