Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: C3aR is highly expressed intracellularly in unstimulated NHEKs and is up-regulated intracellularly as well as on the cell surface of NHEKs upon TLR3 stimulation. NHEKs were stimulated with Poly I:C (10 μg/mL) for 24, 48, and 72 h or left unstimulated. Keratinocytes were stained intracellularly and extracellularly with phycoerythrin-labelled C3aR monoclonal mouse IgG2b antibody. Expression of C3aR was analysed by flow cytometry, and MFIs were calculated. a Eight intracellular staining experiments from 7 different donors are shown. b One representative histogram of intracellular C3aR expression is shown. d Eight extracellular staining experiments from 7 different donors are shown. e One representative histogram of extracellular C3aR expression is shown. The dotted line represents the matched isotype control, the violet line represents the ns cells, and the green line represents the Poly I:C-stimulated cells. c NHEKs were cultured and stimulated with LPS/TLR4 (50 μg/mL), CpG oligonucleotide/TLR9 (2 μg/mL), or Poly I:C/TLR3 (10 μg/mL) for 48 h or left unstimulated, and C3aR expression was analysed intracellularly by flow cytometry. f, g NHEKs were stimulated with Poly I:C for 24 h or left unstimulated. Poly I:C-stimulated cells were washed, and new medium was added. Then, the cells were stimulated with C3a (1 μg/mL) for 24 h or left unstimulated. The supernatants were all collected at the same time point. f The release of C3 was measured by the ELISA technique. g The release of CXCL10 was measured by the ELISA technique. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test (f, g) or by Friedman-Dunn's multiple comparison test selected pairs (a–d), are indicated as follows: +p < 0.05; ++p < 0.01; medians are shown in the graphs. n = 10 experiments from 10 independent donors (c); n = 5 experiments from 5 independent donors (f); n = 7 experiments from 5 independent donors (g). NHEK, normal human epidermal keratinocyte; ns, non-stimulated; MFI, mean fluorescence intensity; C3aR, anaphylatoxin C3a receptor; C3a, active C3 fragment.

Article Snippet: To detect the C3a neo-epitope, which is expressed in activation products and not present in the individual native components, a monoclonal mouse IgG1 antibody (1 μg/mL) from Hycult Biotech, Uden, The Netherlands, was used.

Techniques: Staining, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: TLR3 ligand Poly I:C up-regulates C3 mRNA and protein expression in NHEKs. Intracellular generation of the biologically active fragment of C3, the C3a neo-epitope, is enhanced in parallel via TLR3 in NHEKs. NHEKs were stimulated with Poly I:C (10 μg/mL) for 24 h (C3 mRNA and C3a generation) and for different time points as indicated (C3 protein) or left unstimulated. For a, d, and e, a selective and reversible inhibitor of human CTSL was used to prove if the enhanced generation of C3a neo-epitope is dependent on the presence of CTSL. (a) C3 mRNA expression was analysed by qPCR and tgt/ref ratio was calculated by the [delta] Ct method. Phosphoglycerate kinase 1 was used as reference gene. b Cell-free supernatants were collected after different time periods, and C3 concentrations were determined by ELISA. c–e C3a was stained intracellularly with a monoclonal mouse IgG1 antibody against a specific epitope of this cleavage product of C3 called neo-epitope. The expression of C3a was analysed by flow cytometry, and MFIs were calculated. f One representative histogram of intracellular C3a expression is shown. The dotted line represents the matched isotype control, the violet line represents the ns cells, and the green line represents the Poly I:C-stimulated cells. Significant differences, as determined by the Wilcoxon matched-pairs signed rank test (a, c, d) or by Friedman-Dunn's multiple comparison test selected pairs (b), are indicated as follows: +p < 0.05; ++p < 0.01; +++p < 0.001; medians are shown in graphs. n = 12 experiments from 6 independent donors (a); n = 7 experiments from 7 independent donors (b); n = 7 experiments from 4 independent donors (c); n = 8 experiments from 5 independent donors (d); n = 5 experiments from 4 independent donors (e). NHEK, normal human epidermal keratinocyte;ns, non-stimulated; MFI, mean fluorescence intensity; CTSL, cathepsin L; inh., inhibitor; tgt/ref, target/reference; C3a, active C3 fragment.

Article Snippet: To detect the C3a neo-epitope, which is expressed in activation products and not present in the individual native components, a monoclonal mouse IgG1 antibody (1 μg/mL) from Hycult Biotech, Uden, The Netherlands, was used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Innate Immunity

Article Title: C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

doi: 10.1159/000512547

Figure Lengend Snippet: C3aR, C3 the active fragment of C3, the anaphylatoxin C3a, and the protease CTSL are differentially expressed in normal human epidermis and in NHEKs cultured in low Ca2+ concentrations compared to NHEKs cultured in high Ca2+ concentrations. C3aR expression (rabbit polyclonal, Abcam, Cambridge, UK) (a), C3 expression (rabbit polyclonal, Abcam, Cambridge, UK) (b), C3a expression (monoclonal mouse IgG1, Hycult Biotech, Uden, The Netherlands) (c), and CTSL (monoclonal mouse IgG1, Abcam) (d) expression were detected in the epidermis in skin biopsies of normal human skin by immunohistochemistry. Staining with the corresponding isotypes showed no unspecific binding in the epidermis in skin biopsies of normal human skin by immunohistochemistry. m Mouse IgG1. n Rabbit polyclonal IgGs. One example for each target out of 4 different immunohistochemistry stainings of human skin is shown. NHEKs from different donors were cultured in chamber tek slides in medium containing 0.06 mM CaCL2 (low calcium conditions) and 1.36 mM CaCL2 (high calcium conditions) for 3 days and stained by immunohistochemistry. C3aR expression in high calcium conditions (e), C3aR expression in low calcium conditions (rabbit polyclonal, Abcam, Cambridge, UK) (i), C3 expression in high calcium conditions (f), C3 expression in low calcium conditions (rabbit polyclonal, Abcam, Cambridge, UK) (j), C3a expression in high calcium conditions (g), C3a expression in low calcium conditions (monoclonal mouse IgG1, Hycult Biotech, Uden, The Netherlands) (k), CTSL expression in high calcium conditions (h), CTSL expression in low calcium conditions (monoclonal mouse IgG1, Abcam) (l). Staining with the corresponding isotypes showed no unspecific binding by immunohistochemistry. o Mouse IgG1. p Rabbit polyclonal IgGs. One example for each target and condition out of 4 different immunohistochemistry stainings of NHEKs on chamber tek slides is shown. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; C3aR, anaphylatoxin C3a receptor; C3a, active C3 fragment.

Article Snippet: To detect the C3a neo-epitope, which is expressed in activation products and not present in the individual native components, a monoclonal mouse IgG1 antibody (1 μg/mL) from Hycult Biotech, Uden, The Netherlands, was used.

Techniques: Cell Culture, Expressing, Immunohistochemistry, Staining, Binding Assay

Journal: Stem Cells (Dayton, Ohio)

Article Title: Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

doi: 10.1002/stem.544

Figure Lengend Snippet: Antimicrobial activity of MSC CM is mediated by LL-37 secretion. (A): Synthetic LL-37 showed significant antimicrobial activity against E. coli in a dose-dependent manner. Data are mean ± SD; *, p < .001 versus RPMI by analysis of variance (ANOVA; Bonferroni) n = 3. (B): Synthetic LL-37 displayed dose-dependent antimicrobial effect against P. aeruginosa. Data are mean ± SD; *, p < .001 versus RPMI by ANOVA (Bonferroni) n = 3. Preincubation of MSC CM with anti-LL-37 antibody (1 μ;g/ml), but not with mouse IgG (1 μg/ml), significantly reduced the antimicrobial effect of MSC CM against E. coli (C) and P. aeruginosa (D). Data are mean ± SD; *, p < .0002 versus MSC CM + anti-LL-37 by ANOVA (Bonferroni), n = 5–7. Abbreviations: CFU, colony-forming unit; MSC CM, mesenchymal stem cell conditioned medium; RPMI, RPMI-1640 medium.

Article Snippet: Mouse monoclonal antibody to human LL37/CAP18 clone 3D11 and mouse isotype IgG1 antibody were purchased from Hycult Biotechnology (Netherlands), goat Alexa-Fluor 488-labeled anti-mouse-IgG from Invitrogen, synthetic human LL-37 from AnaSpec (CA) and fetal bovine serum (FBS) from HyClone Laboratories Inc. (Utah).

Techniques: Activity Assay

Journal: Stem Cells (Dayton, Ohio)

Article Title: Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

doi: 10.1002/stem.544

Figure Lengend Snippet: Intratracheal administration of anti-LL 37 antibody reduced the therapeutic effect of MSC in E. coli pneumonia. Coadministration of MSC together with an anti-LL 37 neutralizing antibody (10 μg), but not with mouse IgG isotype antibody, inhibited the therapeutic effect of MSC in bacterial clearance in lung homogenates (A) and BAL fluid (B). Values are mean CFU ± SD; *, p < .002 versus MSC + anti-Ll-37 antibody treated mice; √, p < .005 versus MSC + anti-LL-37 antibody-treated mice by analysis of variance (ANOVA; Bonferroni), n = 5. (C): MSC administration enhanced antimicrobial activity of mouse BAL. BAL samples were incubated with E. coli (105 CFU/ml) for 2 hours, and CFU growth was counted. Data are mean ± SD; *, p < .002 versus BAL of PBS-treated mice by ANOVA (Bonferroni), n = 8–9. Abbreviations: BAL, bronchoalveolar lavage; CFU, colony-forming unit; LH, lung homogenates; MSC, mesenchymal stem cell; PBS, phosphate buffered saline.

Article Snippet: Mouse monoclonal antibody to human LL37/CAP18 clone 3D11 and mouse isotype IgG1 antibody were purchased from Hycult Biotechnology (Netherlands), goat Alexa-Fluor 488-labeled anti-mouse-IgG from Invitrogen, synthetic human LL-37 from AnaSpec (CA) and fetal bovine serum (FBS) from HyClone Laboratories Inc. (Utah).

Techniques: Activity Assay, Incubation