Journal:

Article Title: Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

doi: 10.1073/pnas.0607514104

Figure Lengend Snippet: Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6−/− male mice. WT (open symbols) and D6−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Article Snippet: To block inflammatory chemokines, animals were treated with a mixture of goat antibodies to the mouse CC chemokines CCL3L1 (catalog no. AB450NA), CCL4 (catalog no. AB451NA), CCL2 (catalog no. AB479NA) and rat mAb anti-mouse CCL5 (catalog no. MAB478) purchased lyophilized from R&D Systems, resuspended in PBS, and mixed.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 1. Time course of mechanical hypersensitivity induced by paw incision after intrathecal treatment with anti-chemokine (C-C motif) ligand 2 (CCL2) immunoglobulin G (IgG) (3 ng, dotted bars; 10 ng, gray bars) or control IgG (10 ng, open bars) was examined acutely for several minutes after treatment beginning 1 day after surgical incision (A) and for several days after single treatment beginning 1 day after surgical incision (B). Withdrawal thresholds to von Frey filaments values are expressed as the median (solid line) and 25th and 75th percentiles of 8–14 rats per group. * P 0.05 for comparisons to control IgG values at each time point or # P 0.05 for comparison between 3 ng anti-CCL2– and 10 ng anti- CCL2–treated values at each time point by Kruskal-Wallis analysis of variance on ranks followed by Dunn post hoc test. Black ar- rowhead indicates time point of intrathecal injection.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Control, Comparison, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 2. Representative images of microglial staining with an antibody against ionized calcium-binding adapter molecule 1 (IBA1). Ipsilateral lumbar dorsal spinal cord of rats that underwent sham procedures administered control immunoglobulin G (IgG) (10 ng; A, B, and C) or ipsilateral spinal cord of rats with plantar incision administered 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) or 10 ng control IgG (D, E, and F). Microglial IBA1 immunostaining was increased in the ipsilateral dorsal spinal cord of rats 1 day and 2 days postoperatively (1 dpo and 2 dpo) after plantar incision compared with the levels in rats that underwent sham procedure. A single intrathecal injection of anti-CCL2 IgG (10 ng) administered 1 day after plantar incision reduced the levels of ionized calcium-binding adaptor molecule 1 (IBA1)-immunoreactivity (IR) in the spinal cord of incision rats 24 h after administration (H compared with E) but not 30 min after administration (G compared with D) compared with incision rats that received intrathecal control IgG. Insets depict the morphologic differences in microglia 48 h after incision. Not increased size of microglia in control IgG-treated rats versus anti-CCL2–treated rats (F compared with I). Scale bar in H 150 m and scale bar in I 120 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Staining, Binding Assay, Control, Immunostaining, Injection

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 3. Quantification of ionized calcium-binding adapter molecule 1 (IBA1) immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham-operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immunoglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bonferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Binding Assay, Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 4. Representative images of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) labeling (A, D, and G) and Cd11b (OX42; B, E, and H) in the ipsilateral dorsal spinal cord of sham-operated (A, B, and C) and incision rats treated with 10 ng control immunoglobulin G (IgG) (D, E, and F) or 10 ng anti-chemokine (C-C motif) ligand 2 (CCL2) IgG (G, H, and I) rats 2 days postoperatively (2 dpo) after plantar incision. Rats received a single acute administration of anti-CCL2 IgG (10 ng) on day 1 after plantar incision. High-power confocal images show colocalization of p-p38 MAPK (red) and OX42 (green). Increased levels of cytoplasmic p-p38 MAPK in OX42 immunoreactive microglia of incision rats treated with control IgG compared with sham-operated rats (F compared with C) and incision rats treated with anti-CCL2 IgG (F compared with I). Fluorescent images in panels A, B, D, E, G, and H were inverted in Photoshop (Adobe Systems Inc., San Jose, CA) and converted to grayscale to enhance contrast. Scale bars in G and H 75 m and scale bar in I 5 m.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Labeling, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 5. Quantification of microglial phosphorylated p38 mitogen-ac- tivated protein kinase immunostaining in ipsilateral (ipsi) (gray bar) and contralateral (contra) (open bar) dorsal spinal cord of sham- operated and incision rats 30 min after intrathecal administration of anti-chemokine (C-C motif) ligand 2 (CCL2) (10 ng) or control immu- noglobulin G (IgG) (10 ng) on day 1 postoperatively (1 dpo; A) or 24 h after administration of anti-CCL2 (10 ng) or control IgG (10 ng) on day 2 postoperatively (2 dpo; B). * P 0.05 compared with sham control IgG value within side. # P 0.05 compared with contralateral side within the treatment group. P 0.05 compared with incision control IgG-treated values by two-way analysis of variance with Bon- ferroni post hoc test (n 8 rats per group).

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Immunostaining, Control

Journal: Anesthesiology

Article Title: Contribution of the Chemokine (C-C Motif) Ligand 2 (CCL2) to Mechanical Hypersensitivity after Surgical Incision in Rats

doi: 10.1097/aln.0b013e3181d3d978

Figure Lengend Snippet: Fig. 6. Correlation between spinal markers of microglial activation and behavioral measures of mechanical hypersensitivity 2 days after surgical incision or sham procedure. The association between num- ber of microglial ionized calcium-binding adaptor molecule 1 immu- noreactive (IBA1-IR) pixels (A) or number of microglia positive for phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) (B) and mechanical paw withdrawal thresholds (g) were ex- amined using Spearman rho nonparametric linear regression analysis. CCL2 chemokine (C-C motif) ligand 2; IgG immunoglobulin G.

Article Snippet: Rats that received plantar incision were intrathecally administered CCL2 neutralizing antibody (goat anti-mouse CCL2/MCP-1/LE 15 l at 200 ng/ml or 15 l at 667 ng/ml for 3 and 10 ng total, respectively) or the same amount of goat anti-mouse immunoglobulin G (IgG) (both antibodies from R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Immature forms of human MCPs, e.g. Q 1 -CCL2 display cleavage sites for ApP (1) and DP4 (2). CCL2 can also be processed by MMP-1 (3). All human MCPs are putative substrates of QC/isoQC and they contain in their mature state an N-terminal pE-residue and are protected against aminopeptidase cleavage but not against MMP-1 cleavage. N-terminal degradation of human Q 1 -CCL2 and pE 1 -CCL2 in human plasma monitored using MALDI-TOF mass spectrometry. Internalization of CCR2 from the surface of THP-1 monocytes triggered by CCL2 variants and determined by FACS analysis. (*** p < 0.001 and * p < 0.05 vs. control; ### p < 0.001 and ## p < 0.01 vs. D 3 , ++ p < 0.01 vs. Q 1 , one-way ANOVA followed by Tukey post hoc test, n = 3–8, mean ± SEM). Dependence of THP-1 monocyte migration on the concentration of CCL2(Q 1 -76) and CCL2(pE 1 -76), assessed using a chamber assay and quantification of cells by FACS analysis (*** p < 0.001, ** p < 0.01, Q 1 -CCL2 vs. pE 1 -CCL2, two-way ANOVA followed by Bonferroni post-test, n = 3–7, mean ± SEM).

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: Residue, Clinical Proteomics, Mass Spectrometry, Control, Migration, Concentration Assay, Boyden Chamber Assay

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Specificity constants for the conversion of human and murine CCL2 by human QC/isoQC and murine QC/isoQC

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Analysis of the ratio of total-CCL2 and pE 1 -CCL2 secreted from LPS-stimulated primary cells isolated from QC −/− mice compared to WT littermates (QC +/+ ; n = 4–5, mean ± SEM). Analysis of the ratio of total-CCL2 and pE 1 -CCL2 secreted from LPS-stimulated primary cells isolated from isoQC −/− mice compared to WT littermates (isoQC +/+ ; *** p < 0.001 vs. isoQC +/+ , Student's t -test, n = 4, mean ± SEM). pE 1 -CCL2 formation in serum after peripheral injection of LPS in male QC −/− mice compared to WT littermates (QC +/+ ; n = 6–7, mean ± SEM). pE 1 -CCL2 formation in serum after peripheral injection of LPS in male isoQC −/− mice, compared to WT littermates (QC +/+ ; *** p < 0.001 vs. isoQC +/+ +LPS, Student's t -test, n = 5–8, mean ± SEM). IsoQC-deficiency leads to impaired monocyte recruitment to lungs after intranasal LPS-application in isoQC −/− compared to WT littermates (isoQC +/+ ; * p < 0.05 vs. isoQC +/+ +LPS, Student's t -test, n = 7–8, mean ± SEM). Analysis of neutrophils in bronchoalveolar fluid after intranasal LPS-application in isoQC −/− compared to WT littermates (isoQC +/+ ; n = 7–8, mean ± SEM).

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: Isolation, Injection

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Representative FACS analysis showing the infiltrating monocyte population in QC +/+ and QC −/− mice dissected by specific staining. Quantification of infiltrating monocytes and granulocytes in QC +/+ mice (black bars) and QC −/− mice (open bars; n = 8–13, mean ± SEM). Lavage fluid from ( B ) was analyzed for total-CCL2 (black bars) and pE 1 -CCL2 (open bars; n.s., not significant, Student's t -test, mean ± SD). Representative FACS analysis showing the infiltrating monocyte population in isoQC +/+ and isoQC −/− mice. Quantification of infiltrating monocytes and granulocytes in isoQC +/+ mice (black bars) and isoQC −/− mice (open bars; *** p < 0.001 vs. isoQC +/+ Thio, Student's t -test, n = 10–14, mean ± SEM). Lavage fluid from ( E ) was analyzed for total-CCL2 (black bars) and pE 1 -CCL2 (open bars; *** p < 0.001 vs. pE 1 -CCL2 from isoQC +/+ mice, Student's t -test, mean ± SD).

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Analysis of total-CCL2 (black bars) and pE 1 -CCL2 (open bars) after application of varying doses PQ529 to LPS-stimulated primary murine glia cells isolated from C57BL/6J WT mice compared to unstimulated controls (*** p < 0.001 vs. pE 1 -CCL2 (0 µM PQ529), ANOVA followed by Tukey post hoc test, n = 3–4, mean ± SEM). Analysis of CCL2 gene expression in LPS-stimulated primary glia cells derived from (* p < 0.05, ** p < 0.01 vs. PQ529 0 µM, ANOVA followed by Tukey post hoc test, n = 3–4, mean ± SEM). Representative FACS image showing the reduction of infiltrating monocytes after application of PQ529 (30 mg/kg, i.p.). Dose-dependent reduction of infiltrating monocytes in absence (black bars) or presence (red bars) of intraperitoneal PQ529 treatment (** p < 0.01 vs. Thio (+), ANOVA followed by Tukey post hoc test, n = 5–6, mean ± SEM, female mice). Inhibition of monocyte infiltration after oral application of PQ50 (red bars) and PQ529 (white bar; ** p < 0.01 vs. Thio (+), ANOVA followed by Tukey post hoc test, n = 5–6, mean ± SEM, female mice).

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: Isolation, Gene Expression, Derivative Assay, Inhibition

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: Monocyte adhesion and total adhering cells 2 days after cuff placement in absence (black bars) or presence (open bars) of PQ50 (QCI) treatment (* p < 0.05 vs. control, Student's t -test, n = 5, mean ± SD). In addition, the CCL2-positive area was calculated in cross-sections within the media and neointima in absence (black bars) and presence (open bars) of PQ50 (QCI) treatment (** p < 0.01, *** p < 0.001 vs. control. Student's t -test, n = 5, mean ± SD). Morphometric analysis of cuffed vessel segments shows a reduction in the degree of lumen stenosis (* p < 0.05 vs. control, Student's t -test, n = 10, mean ± SD). Inset: Example for lumen stenosis after 2 weeks (black arrows). Neointima formation and media thickness (inset) of the cuffed vessel segments of mice sacrificed after 2 weeks, treated in absence (black bars) and presence (open bars) of QC-inhibitor PQ50 (QCI; * p < 0.05 vs. control, Student's t -test, n = 10, mean ± SD).

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: Control

Journal: EMBO Molecular Medicine

Article Title: The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

doi: 10.1002/emmm.201100158

Figure Lengend Snippet: IsoQC is the major enzyme for maturation of CCL2 in vivo ensuring stability and receptor activation leading to monocyte migration. Application of small molecules, which inhibit isoQC provokes the secretion of immature forms (Q 1 -CCL2) prone to aminopeptidase cleavage generating truncated forms (}-CCL2), devoid of bioactivity under physiological conditions.

Article Snippet: For the detection of murine total-CCL2, goat-anti mouse CCL2 (Santa Cruz) was applied in combination with anti-goat-IgG-HRP conjugate (R&D Systems).

Techniques: In Vivo, Activation Assay, Migration