Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: In vitro citrullination of bacterially produced chemokines leads to their partial degradation. ( a ) Colloidal Coomassie stained SDS-PAGE analysis of the MCP-1/CCL2 products of in vitro citrullination. In vitro citrullination of commercially available recombinant MCP-1/CCL2 and self-made bacterially produced MCP-1/CCL2 performed with recombinant human PAD2 and PAD4 or rabbit PAD2, respectively. Colloidal Coomassie stained MCP-1 bands are shown while recombinant humanPAD2/4 or rabbit PAD2 (Sigma Aldrich/Merck, Waltham, MA, USA) were added to reactions into the quantities below the Colloidal Coomassie sensitivity limits and cannot be visualized. ( b ) Immunoblot analysis of bacterially produced MCP-1/CCL2 upon an in vitro citrullination reaction. Self-made full-length bacterially produced MCP-1/CCL2 was citrullinated in vitro with rabbit PAD2 and resolved on SDS-PAGE, stained with Ponceau S. detection of total MCP-1/CCL2 and modified citrullines with Senshu’s antibody that recognizes that modified citrullines were made according to Senshu’s protocol . Results shown are a representative of three or more repetitive experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: In Vitro, Produced, Staining, SDS Page, Recombinant, Western Blot, Modification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Mass-spectrometry verification of successful in vitro citrullination of MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 (mature form of the protein spans residues 24–99). The first row shows 23 amino acid-long signaling peptide and the second row–mature protein of 76 residues. All arginine residues are highlighted with circles. The boxed areas show the peptide that contains the citrullinated arginine residues that were detected. ( b ) Annotated tandem mass spectrometry (MS/MS) fragmentation spectrum for the citrullinated MCP1/CCL2 peptide, showing the citrullinated arginine residue at position 3 of the studied peptide (R45). The MS/MS fragmentation data were annotated using Expert System (Max Planck Institute of Biochemistry). The precursor ion was observed with a mass error of 1 part per million, and the error for the fragment ions was 0.02 daltons.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Mass Spectrometry, In Vitro, Sequencing, Tandem Mass Spectroscopy, Residue

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Detection of citrullinated recombinant human MCP-1/CCL2. Standard curves of citrullinated recombinant human MCP-1/CCL2 were set up using enzyme-linked immunosorbent assay in duplicate ( a ) standard curve of citrullinated E. coli produced recombinant human MCP-1/CCL2 chemokine (R&D Systems), ( b ) standard curve for citrullinated MCP-1/CCL2 chemokine produced by and purified from human HEK 293T cells. Absorbance at 450 nm is shown. Coefficients of determination R 2 are indicated in red on the relevant plots. Results shown are a representative of three or more repetitive experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Purification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Site-directed mutagenesis of potential glycosylation site at asparagine-14 in mammalian cell-produced MCP-1 significantly destabilizes recombinant human MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 with indicated positions of predicted N-glycosylation and citrullination site confirmed by a mass-spectrometry. ( b ) Immunoblot analysis of bacterially versus mammalian cell-produced MCP-1/CCL2 upon an in vitro citrullination reaction. Bacterially produced wild-type MCP-1/CCL2 or HEK293T cell-produced wild-type and N14Q mutant version of chemokine were incubated with EDTA-free proteinase inhibitor cocktail supplemented cellular lysates prepared from the control (vehicle-transfected) or indicated hPAD enzyme-transfected HEK293T cells. Citrullination reactions that were performed directly within cellular lysates essentially as published before . Recombinant chemokine was concentrated with immunoprecipitation and resolved on 10–18% polyacrylamide gradient SDS-PAGE. Results shown are a representative of three experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Mutagenesis, Produced, Recombinant, Sequencing, Mass Spectrometry, Western Blot, In Vitro, Incubation, Transfection, Immunoprecipitation, SDS Page

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Lung samples were removed at days 35 and 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice, and all soluble proteins were measured by specific ELISA. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with appropriate the C.B-17SCID/bg group that received normal fibroblasts. ττP ≤ 0.01, τττP ≤ 0.001 compared with whole-lung cytokine and chemokine levels at the day 35 time point in the C.B-17SCID/bg groups with either IPF/UIP or NSIP fibroblasts The whole-lung cytokine and chemokine levels in control C.B-17SCID/bg group that did not receive fibroblasts were as follows: IL-13, 0.18 ± 0.014 ng/mg protein; CCL6, 0.37 ± 0.05 ng/mg protein; and CCL21, 2.0 ± 0.1 ng/mg protein.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: SuperArray Analysis of Human CCR7 and CCL21 and TaqMan Analysis of Human CCR7 in Whole-Lung Samples from C.B-17SCID/bg Mice at Day 35 after i.v. Human Fibroblast Injection

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Injection

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Representative Mason trichrome-stained histological sections from C.B-17SCID/bg mice that received normal (A–C), NSIP (D–F), or IPF/UIP (G–I) fibroblasts. No interstitial remodeling was apparent in C.B-17SCID/bg mice that received normal fibroblasts, but vascular anomalies were observed in this group (B), and the IgG (A), anti-CCL21 monoclonal antibody (B), and anti-CCR7 monoclonal antibody (C) therapies did not alter the lung histological appearance in this group. Pulmonary remodeling was apparent in C.B-17SCID/bg mice that received NSIP fibroblasts, and this pattern was not altered by IgG (D), whereas the anti-CCL21 antibody (E) or anti-CCR7 antibody (F) therapies markedly reduced the interstitial remodeling in whole-lung samples. Interstitial pulmonary fibrosis was apparent in C.B-17SCID/bg mice that received IPF/UIP fibroblasts, and this pattern was not altered by IgG (G), whereas the anti-CCL21 antibody (H) or anti-CCR7 antibody (I) therapies markedly reduced the interstitial remodeling in whole-lung samples. Monoclonal antibody therapies began at day 35 and continued to day 63, and lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Original magnification, ×400.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Staining

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Quantitative TaqMan PCR analysis of extracellular matrix-associated genes MMP-2 (top) and MMP-19 (bottom) in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts. Changes in gene expression are expressed as mean ± SEM of the fold increase in transcript expression above a group of C.B-17SCID/bg mice that received PBS, PKH26, and one of IgG, anti-CCL21 antibody, and anti-CCR7 antibody. *P ≤ 0.05, ***P ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Expressing

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung hydroxyproline levels in C.B-17SCID/bg mice that received no fibroblasts (ie, control) or received normal, NSIP, or IPF/UIP fibroblasts. All groups of mice received either IgG or monoclonal antibody therapy. IgG, anti-CCL21, and anti-CCR7 monoclonal antibody therapies began in separate groups of C.B-17SCID/bg mice at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Data shown are mean ± SEM. *P ≤ 0.05 compared with the control C.B-17SCID/bg group, which did not receive any human fibroblasts; τττP ≤ 0.001 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgG treatment; τP ≤ 0.05 compared with the appropriate C.B-17SCID/bg group that received human fibroblasts and IgC treatment.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Control

Journal:

Article Title: Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

doi: 10.2353/ajpath.2007.060649

Figure Lengend Snippet: Whole-lung murine IL-13, CCL6, and CCL21 levels in C.B-17SCID/bg mice that received normal, NSIP, or IPF/UIP fibroblasts and IgG or monoclonal antibody therapies. Anti-CCL21 and anti-CCR7 monoclonal antibody therapies began at day 35 and continued to day 63. Lung samples were removed at day 63 after the adoptive i.v. transfer of human fibroblasts into C.B-17SCID/bg mice. Specific ELISA was used to measure all soluble proteins. Data shown are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 compared with indicated protein levels measured in whole-lung samples from C.B-17SCID/bg mice that received human fibroblasts.

Article Snippet: Thirty-five days later, all groups of five C.B-17SCID/bg mice received mouse IgG, mouse anti-human CCL21 monoclonal antibody, or mouse anti-human CCR7 monoclonal antibody (all at 10 μg/ml; R&D Systems, Minneapolis, MN) every other day from days 35 to 63.

Techniques: Enzyme-linked Immunosorbent Assay