Journal: Developmental biology

Article Title: Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

doi: 10.1016/j.ydbio.2011.07.021

Figure Lengend Snippet: Fig. 1. Concomitant MMP9 and uPA gene-deficiency exaggerates the effect observed in single-deficient mice on bone growth. (A–B) Cohorts of male and female mice are presented in the left and right panel, respectively. (A) Survival data are shown for a cohort of wild-type, MMP9-deficient, uPA-deficient, and MMP9;uPA double-deficient mice. The total number of mice (n) in each group is indicated together with the number of mice that died during the 22 week observation period in brackets. (B) Plot of weight versus age of the mice in the cohorts in A. (C) X-ray pictures of 26 weeks old male wild-type, MMP9-deficient, uPA-deficient, and MMP9;uPA double-deficient mice. (D) The mean bone length of tibia, isolated from wild-type (n=14♂; 13♀), MMP9-deficient (n=14♂; 10♀), uPA-deficient (n=7♂; 12♀), and MMP9;uPA double-deficient (n=11♂; 4♀) mice at age 26 weeks is presented as horizontal lines in the scatter plots.

Article Snippet: Sections were treated with proteinase K (5 μg/ml) for 15 min at 37 °C and incubated with a rabbit polyclonal antibody against murine MMP9 (Ab38898; Abcam, Cambridge, UK) and detected with EnVisionTM HRP rabbit reagent (K4003, Dako, Glostrup, Denmark) as described in (Rasch et al., 2010).

Techniques: Isolation

Journal: Developmental biology

Article Title: Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

doi: 10.1016/j.ydbio.2011.07.021

Figure Lengend Snippet: Fig. 2. Different healing kinetics of full-thickness skin wounds in 3 cohorts of protease-deficient mice. (A–D) Wound healing in wild-type, MMP9-deficient, uPA-deficient, and MMP9;uPA double-deficient mice (left column), in wild-type, MMP9-deficient, tPA-deficient, and MMP9;tPA double-deficient mice (middlecolumn), and in wild-type,MMP9-deficient, uPAR-deficient, and MMP9;uPAR double-deficient mice(right column)arepresented. (A)Representativeexamplesofwound repairprogression(days7,14, and21)inthe threedifferent cohorts.(B)The percentage of healed wounds in mice plotted versus time after wound incision. A significant delay is observed in MMP9;uPA double-deficient mice and a modest delay in both groups of mice with single deficiencies is seen, as compared to wild-type mice. (C) The average wound length is plotted as a function of time after incision. The wound length of MMP9;uPA double-deficient mice was significantly increased compared to both wild-type and MMP9 or uPA single-deficient mice. The average wound length of wild-type, tPA-deficient, and uPAR-deficient mice was in contrast indistinguishable during re-epithelialization. The symbol and number of mice are shown in panels B and C for each cohort. (D) Statistical analysis of each cohort.

Article Snippet: Sections were treated with proteinase K (5 μg/ml) for 15 min at 37 °C and incubated with a rabbit polyclonal antibody against murine MMP9 (Ab38898; Abcam, Cambridge, UK) and detected with EnVisionTM HRP rabbit reagent (K4003, Dako, Glostrup, Denmark) as described in (Rasch et al., 2010).

Techniques:

Journal: Developmental biology

Article Title: Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

doi: 10.1016/j.ydbio.2011.07.021

Figure Lengend Snippet: Fig. 3. Histological analysis of wounds in wild-type, MMP9-deficient, uPA-deficient, MMP9;uPA double-deficient mice at days 7 and 14 after incision. Upper panels: H&E-stained sections of wounds isolated from the indicated mice at day 7 and day 14 post-wounding. Images show representative examples of normal appearing non-injured skin with dermis (D), wound site with newly formed epidermis (E), provisional matrix (*), and the granulation tissue (GT) at low magnification (a–d and i–l) with inserted frames showing the edge of the keratinocyte (K) tongue presented in high magnification (e–h and m–p). Arrow heads indicate the wound edge (a, b, c, d), and arrows mark the keratinocytes (e–h and m–p). On day 14, all wild-type mice possessed a fully closed wound in contrast to some of the uPA-deficient and MMP9-deficient mice and all double-deficient mice. Note the wound edge of the double-deficient mice, showing epidermal sprouts into granulation tissue (p, arrows). Bars: ~280 μm (a–d and i–l), ~35 μm (e–h and m–o), and ~140 μm (p). Lower panels: Morphometric analysis of keratinocyte re-epithelialization in wounds. Scatter plot of the relative re-epithelialization of keratinocytes at day 7 post-wounding (left panel: 12 wild- type mice, 8 MMP9-deficient, 11 uPA-deficient, and 7 MMP9;uPA double-deficient mice) and at day 14 post-wounding (right panel: 10 wild-type mice, 10 MMP9-deficient, 13 uPA- deficient, and 8 MMP9;uPA double-deficient mice). The relative migration was determined as the migration distance from the wound edge to the front of the leading-edge keratinocytes, divided by the distance between the two wound edges.

Article Snippet: Sections were treated with proteinase K (5 μg/ml) for 15 min at 37 °C and incubated with a rabbit polyclonal antibody against murine MMP9 (Ab38898; Abcam, Cambridge, UK) and detected with EnVisionTM HRP rabbit reagent (K4003, Dako, Glostrup, Denmark) as described in (Rasch et al., 2010).

Techniques: Staining, Isolation, Migration

Journal: Developmental biology

Article Title: Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

doi: 10.1016/j.ydbio.2011.07.021

Figure Lengend Snippet: Fig. 4. Gene-specific compensatory upregulation of uPA in MMP9-deficient mice during skin wound healing. (A) In situ hybridization of tissue sections from 7 day old skin wounds for uPA mRNA in wild-type (a) and MMP9-deficient mice (b); immunohis- tochemical staining for MMP9 on sections of wounds from wild-type (c) and uPA- deficient mice (d). Distinct expression of uPA mRNA is seen in leading-edge keratinocytes (K) in wild-type (a, arrows) and MMP9-deficient mice (b, arrows). Similarly, expression of MMP9 immunoreactivity was observed in wild-type and uPA- deficient mice in leading-edge keratinocytes (c and d, respectively, arrows). Bars: ~70 μm (a–b) and ~35 μm (c–d). (B) Fibrin overlay zymography of pooled wound extracts prepared from 3 mice with 7 day old wounds and as control a mixture of purified human pKal, murine uPA, and human tPA. In the absence (upper) or presence of the monoclonal antibody mU1 (65 μg/ml), blocking the function of mouse uPA (lower). (C) Gelatin substrate zymography of pooled wound extracts from 3 mice prepared from 7 day old wounds were analyzed in the absence (upper) or presence of 10 mM EDTA (lower) to block MMP activity. (D) Western blot of wound extracts prepared from 7 day old wounds obtained from 3 uPA-deficient, 3 MMP9-deficient, and 3 wild-type mice were analyzed under non-reducing conditions. Similar amount of pro- uPA/uPA as indicated by the arrow was seen in wild-type and MMP9-deficient mice. Loading control of the wound lysates by use of β-actin is presented in the lower panel. The position of the molecular weight marker (kDa) is indicated at the right.

Article Snippet: Sections were treated with proteinase K (5 μg/ml) for 15 min at 37 °C and incubated with a rabbit polyclonal antibody against murine MMP9 (Ab38898; Abcam, Cambridge, UK) and detected with EnVisionTM HRP rabbit reagent (K4003, Dako, Glostrup, Denmark) as described in (Rasch et al., 2010).

Techniques: In Situ Hybridization, Staining, Expressing, Zymography, Control, Blocking Assay, Activity Assay, Western Blot, Molecular Weight, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Human PMNs were activated at 37°C with PAF at 10−7 M for 15 min followed by fMLP at 10−7 M for 30 min. Cells were double immunostained with Alexa 546 and murine anti-MMP-9 IgG (A, second panel), or murine anti-human MMP-8 IgG (B, second panel) or non-immune murine (Ms) IgG (C, second panel) and with Alexa 488 and rabbit anti-TIMP-1 IgG (A and B third panels) or non-immune rabbit (Rb) IgG (C, third panel). The anti-MMP-8 and anti-MMP-9 IgGs used recognize both pro and active forms of these MMPs. Cells were examined using a Normarski objective (A-C, left panels) and co-localization of MMPs and TIMP-1 on the surface of the activated PMNs was assessed by confocal microscopy (see merged images in the right panels for A-C). The white bars are 5 microns in length. Note the strong co-localization of TIMP-1 with both MMP-8 and MMP-9 on the surface of activated PMNs. The results shown are representative of at least 3 different PMN preparations.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Confocal Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from Mmp-8−/− x Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP to induce surface expression of Timp-1. In A, the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B, the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B, images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A) or pro and active Mmp-8 (in B) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Expressing, Incubation, Software, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from WT, Timp-1−/−, Mmp-8−/−, and Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP. The cells were then fixed and immunostained for surface-bound Mmp-8 (in A) or surface-bound Mmp-9 (in B) using antibodies that detect both pro and active forms of these Mmps. Surface levels of Mmp-8 or Mmp-9 were quantified as described in Methods and expressed as a percentage of the amount of Mmp-8 or Mmp-9 respectively, that were expressed on the surface of activated WT PMNs. Data are mean + SEM from 3–4 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs. In C-E, PMNs from WT, Timp-1−/−, and Mmp-8−/− and or Mmp-9−/− mice were activated and fixed as described in A. Equal numbers of cells from each genotype were pre-incubated for 1 h at 37°C in triplicate with and without 1–10-phenanthroline (a non-selective MMP inhibitor). Cells were then incubated at 37°C with: 1) type I-collagen conjugated to quenched FITC for 18 h (in C); 2) gelatin conjugated to quenched FITC for 6 h (in D); or 3) McaPLGLDpaAR (a quenched fluorogenic peptide substrate which is cleaved by both Mmp-8 and Mmp-9) for 3 h (in E). Mmp-mediated cleavage of each substrate was quantified in cell-free supernatant samples by fluorimetry as the 1,10-phenanthroline-inhibitable cleavage of the substrate, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate by WT cells from 4–5 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Two Tailed Test, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Mmp-9 protein levels contained within and released by WT and Timp-1 −/− PMNs

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Extracts of freshly-isolated unstimulated PMNs from WT and Timp-1−/− mice and cell-free supernatant fluids from WT and Timp-1−/− PMN that had been activated with 10−6 M fMLP for 30 min were immuno-blotted for Mmp-9 (A) or Mmp-8 (B). The arrows indicate the proMMP-9 and proMmp-8 forms of the Mmps. Note that extracts of unstimulated WT and Timp-1−/− PMNs contain similar amounts of proMmp-9 and proMmp-8 (Mr = 100 kDa for Mmp-9 and 85 kDa for Mmp-8). In addition, when activated with fMLP, Timp-1−/− PMNs freely release similar amounts of Mmp-8 and Mmp-9 exclusively as the latent proMmp forms.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated PMNs from Mmp-9−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-9, and then immunostained with rabbit anti-Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488. In B, PAF- and fMLP-activated PMNs from Mmp-8−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-8, and then immunostained with rabbit anti-Mmp-8 IgG, or non-immune rabbit IgG followed by goat- anti-rabbit F(ab)2-conjugated to Alexa 488. In A-B, the antibodies used detect both the pro and active forms of the Mmps. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with cells incubated without exogenous murine proMmp-9 or proMmp-8. In C, Timp-1−/− PMNs were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-9 for 1 h; 3) 200 nM Timp-1 for 1 h and then with proMmp-9 for 1 h; 4) 200 nM Timp-2 for 1 h, and then with proMmp-9 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in buffer. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-9. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In D, Timp-1−/− PMN were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-8 for 1 h; 3) 200 nM Timp-1 for 1 h and then proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then proMmp-8 for 1 h; 5) 200 nM myeloperoxidase for 1 h and then 200 nM proMmp-8 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in PBS. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-8. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In A-D, the results shown are representative of at least 3 independent experiments. In E-F, equal numbers of PAF- and fMLP-activated PMNs from Timp-1−/− mice were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and 200 nM proMmp-9 or proMmp-8 alone for 1 h; 3) 200 nM Timp-1 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; The cells were fixed and then incubated at 37°C with: 1) gelatin conjugated to quenched FITC for 18 h (in E); or 2) type-I collagen conjugated to quenched FITC for 18 h (in F). In E-F, Mmp-mediated cleavage of each substrate in the presence of amino-phenyl mercuric acetate was quantified in cell-free supernatant samples using fluorimetry, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate in cells incubated without exogenous proteins; n=4–5 separate experiments. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. Asterisks indicate P < 0.05 compared with all other groups.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated Mmp-8−/− x Mmp-9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length proMmp-8 protein (FL Mmp8) or 400 nM mutant proMmp-8 protein lacking the COOH-terminal hemopexin domain (MT Mmp-8). Bound Mmp-8 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-8 (which is present in both the FL and MT forms of Mmp-8). In B, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length Mmp-9 (FL Mmp9) or 400 nM mutant Mmp-9 lacking the COOH-terminal hemopexin domain (MT Mmp9). Bound Mmp-9 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-9 (which is present in both the FL and MT forms of Mmp-8 and Mmp-9). In A-B, Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the no exogenous Mmp group or the group indicated. In C-D, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated at 4°C for 60 min with or without 500 nM soluble murine hemopexin protein. Cells were then incubated at 4°C for 60 min with or without 400 nM exogenous proMmp-8 or 400 nM exogenous proMmp-9. Mmp-8 and Mmp-9 that bound to cells was detected by immunostaining the cells with Alexa-488 for Mmp-8 (C) or Mmp-9 (D), as described above. Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the group incubated without exogenous proteins or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Mutagenesis, Immunostaining, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, human PMNs were incubated with 1 μg/mL LPS for 4 h at 37°C to induce NET formation, or without LPS as a control. In A, the cells were fixed and then double immunostained with a green fluorophore for TIMP-1 (second panels) and a red fluorophore for MMP-8 or MMP-9 (third panels). Nuclei were counterstained blue with 4’,6-diamidino-2-phenylindole (DAPI). Co-localization of TIMP-1 and MMPs in PMN-associated NETs was assessed by confocal microscopy (see merged images in the fourth panels). The results shown are representative of 4 different PMN preparations. In B, NET induction by LPS was quantified by staining the samples for extracellular DNA with SYTOX™ Blue Nucleic Acid Stain, and quantifying the staining as described in Methods. Data are mean + SEM; n = 4 experiments. Data were analyzed using the Mann-Whitney U tests. Asterisk indicates P < 0.05 compared with unstimulated cells.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Control, Confocal Microscopy, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: NHPs were exposed to air (n = 4; white bars) or smoke (n = 6; gray bars) for 4 weeks, and PMNs were isolated from peripheral blood samples from each animal both at baseline and after 4 weeks of air or CS exposure. Cells were double immunostained with Alexa 488 and murine anti-TIMP-1 IgG or non-immune murine (Ms) IgG (A and B second panels) and with Alexa 546 and rabbit anti-MMP-8 IgG or rabbit anti-MMP-9 IgG (A and B third panels). Nuclei in the cells were counterstained blue using 4’,6-diamidino-2-phenylindole (DAPI, A and B first panels). Co-localization of TIMP-1 and MMPs on the surface of PMNs was assessed using confocal microscopy (see merged images in the fourth panels in A and B). The white bars are 5 microns in length. In C-E, surface TIMP-1 or MMPs levels were quantified using image analysis software, as described in Methods. Data are mean + SEM; n = 4 air-exposed NHPs and n = 6 smoke-exposed NHPs (200–500 cells were analyzed per animal). In C-E, data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with baseline or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Isolation, Confocal Microscopy, Software, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Human PMNs were activated at 37°C with PAF at 10−7 M for 15 min followed by fMLP at 10−7 M for 30 min. Cells were double immunostained with Alexa 546 and murine anti-MMP-9 IgG (A, second panel), or murine anti-human MMP-8 IgG (B, second panel) or non-immune murine (Ms) IgG (C, second panel) and with Alexa 488 and rabbit anti-TIMP-1 IgG (A and B third panels) or non-immune rabbit (Rb) IgG (C, third panel). The anti-MMP-8 and anti-MMP-9 IgGs used recognize both pro and active forms of these MMPs. Cells were examined using a Normarski objective (A-C, left panels) and co-localization of MMPs and TIMP-1 on the surface of the activated PMNs was assessed by confocal microscopy (see merged images in the right panels for A-C). The white bars are 5 microns in length. Note the strong co-localization of TIMP-1 with both MMP-8 and MMP-9 on the surface of activated PMNs. The results shown are representative of at least 3 different PMN preparations.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Confocal Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from Mmp-8−/− x Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP to induce surface expression of Timp-1. In A, the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B, the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B, images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A) or pro and active Mmp-8 (in B) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Expressing, Incubation, Software, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, PMNs from WT, Timp-1−/−, Mmp-8−/−, and Mmp-9−/− mice were activated for 15 min at 37°C with 10−6 M PAF followed by 10−6 M fMLP. The cells were then fixed and immunostained for surface-bound Mmp-8 (in A) or surface-bound Mmp-9 (in B) using antibodies that detect both pro and active forms of these Mmps. Surface levels of Mmp-8 or Mmp-9 were quantified as described in Methods and expressed as a percentage of the amount of Mmp-8 or Mmp-9 respectively, that were expressed on the surface of activated WT PMNs. Data are mean + SEM from 3–4 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs. In C-E, PMNs from WT, Timp-1−/−, and Mmp-8−/− and or Mmp-9−/− mice were activated and fixed as described in A. Equal numbers of cells from each genotype were pre-incubated for 1 h at 37°C in triplicate with and without 1–10-phenanthroline (a non-selective MMP inhibitor). Cells were then incubated at 37°C with: 1) type I-collagen conjugated to quenched FITC for 18 h (in C); 2) gelatin conjugated to quenched FITC for 6 h (in D); or 3) McaPLGLDpaAR (a quenched fluorogenic peptide substrate which is cleaved by both Mmp-8 and Mmp-9) for 3 h (in E). Mmp-mediated cleavage of each substrate was quantified in cell-free supernatant samples by fluorimetry as the 1,10-phenanthroline-inhibitable cleavage of the substrate, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate by WT cells from 4–5 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Two Tailed Test, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: Extracts of freshly-isolated unstimulated PMNs from WT and Timp-1−/− mice and cell-free supernatant fluids from WT and Timp-1−/− PMN that had been activated with 10−6 M fMLP for 30 min were immuno-blotted for Mmp-9 (A) or Mmp-8 (B). The arrows indicate the proMMP-9 and proMmp-8 forms of the Mmps. Note that extracts of unstimulated WT and Timp-1−/− PMNs contain similar amounts of proMmp-9 and proMmp-8 (Mr = 100 kDa for Mmp-9 and 85 kDa for Mmp-8). In addition, when activated with fMLP, Timp-1−/− PMNs freely release similar amounts of Mmp-8 and Mmp-9 exclusively as the latent proMmp forms.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated PMNs from Mmp-9−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-9, and then immunostained with rabbit anti-Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)2-conjugated to Alexa 488. In B, PAF- and fMLP-activated PMNs from Mmp-8−/− and Timp-1−/− mice were incubated with or without 50–400 nM exogenous murine proMmp-8, and then immunostained with rabbit anti-Mmp-8 IgG, or non-immune rabbit IgG followed by goat- anti-rabbit F(ab)2-conjugated to Alexa 488. In A-B, the antibodies used detect both the pro and active forms of the Mmps. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with cells incubated without exogenous murine proMmp-9 or proMmp-8. In C, Timp-1−/− PMNs were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-9 for 1 h; 3) 200 nM Timp-1 for 1 h and then with proMmp-9 for 1 h; 4) 200 nM Timp-2 for 1 h, and then with proMmp-9 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in buffer. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-9. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In D, Timp-1−/− PMN were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and then 200 nM proMmp-8 for 1 h; 3) 200 nM Timp-1 for 1 h and then proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then proMmp-8 for 1 h; 5) 200 nM myeloperoxidase for 1 h and then 200 nM proMmp-8 for 1 h. Unbound proteins were removed after each incubation step by washing PMN twice in PBS. Non-permeabilized PMNs were then immunostained with Alexa 488 for surface-bound pro and active Mmp-8. Data are mean + SEM; n = 150–200 cells per group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with all other groups. In A-D, the results shown are representative of at least 3 independent experiments. In E-F, equal numbers of PAF- and fMLP-activated PMNs from Timp-1−/− mice were incubated at 4°C with: 1) no exogenous proteins for 2 h; 2) no exogenous proteins for 1 h and 200 nM proMmp-9 or proMmp-8 alone for 1 h; 3) 200 nM Timp-1 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; 4) 200 nM Timp-2 for 1 h and then 200 nM proMmp-9 or proMmp-8 for 1 h; The cells were fixed and then incubated at 37°C with: 1) gelatin conjugated to quenched FITC for 18 h (in E); or 2) type-I collagen conjugated to quenched FITC for 18 h (in F). In E-F, Mmp-mediated cleavage of each substrate in the presence of amino-phenyl mercuric acetate was quantified in cell-free supernatant samples using fluorimetry, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate in cells incubated without exogenous proteins; n=4–5 separate experiments. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. Asterisks indicate P < 0.05 compared with all other groups.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A, PAF- and fMLP-activated Mmp-8−/− x Mmp-9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length proMmp-8 protein (FL Mmp8) or 400 nM mutant proMmp-8 protein lacking the COOH-terminal hemopexin domain (MT Mmp-8). Bound Mmp-8 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-8 (which is present in both the FL and MT forms of Mmp-8). In B, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated for 2 h at 4°C with or without 400 nM full length Mmp-9 (FL Mmp9) or 400 nM mutant Mmp-9 lacking the COOH-terminal hemopexin domain (MT Mmp9). Bound Mmp-9 was detected by immunostaining cells with Alexa 488 using an antibody raised to the hinge region of Mmp-9 (which is present in both the FL and MT forms of Mmp-8 and Mmp-9). In A-B, Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the no exogenous Mmp group or the group indicated. In C-D, PAF- and fMLP-activated Mmp-8−/− x Mmp9−/− PMNs were incubated at 4°C for 60 min with or without 500 nM soluble murine hemopexin protein. Cells were then incubated at 4°C for 60 min with or without 400 nM exogenous proMmp-8 or 400 nM exogenous proMmp-9. Mmp-8 and Mmp-9 that bound to cells was detected by immunostaining the cells with Alexa-488 for Mmp-8 (C) or Mmp-9 (D), as described above. Data are mean + SEM; n = 3 separate experiments each analyzing 300 cells/group. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.05 compared with the group incubated without exogenous proteins or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Mutagenesis, Immunostaining, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: In A and B, human PMNs were incubated with 1 μg/mL LPS for 4 h at 37°C to induce NET formation, or without LPS as a control. In A, the cells were fixed and then double immunostained with a green fluorophore for TIMP-1 (second panels) and a red fluorophore for MMP-8 or MMP-9 (third panels). Nuclei were counterstained blue with 4’,6-diamidino-2-phenylindole (DAPI). Co-localization of TIMP-1 and MMPs in PMN-associated NETs was assessed by confocal microscopy (see merged images in the fourth panels). The results shown are representative of 4 different PMN preparations. In B, NET induction by LPS was quantified by staining the samples for extracellular DNA with SYTOX™ Blue Nucleic Acid Stain, and quantifying the staining as described in Methods. Data are mean + SEM; n = 4 experiments. Data were analyzed using the Mann-Whitney U tests. Asterisk indicates P < 0.05 compared with unstimulated cells.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Incubation, Control, Confocal Microscopy, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Promotes PMN Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces

doi: 10.4049/jimmunol.1801466

Figure Lengend Snippet: NHPs were exposed to air (n = 4; white bars) or smoke (n = 6; gray bars) for 4 weeks, and PMNs were isolated from peripheral blood samples from each animal both at baseline and after 4 weeks of air or CS exposure. Cells were double immunostained with Alexa 488 and murine anti-TIMP-1 IgG or non-immune murine (Ms) IgG (A and B second panels) and with Alexa 546 and rabbit anti-MMP-8 IgG or rabbit anti-MMP-9 IgG (A and B third panels). Nuclei in the cells were counterstained blue using 4’,6-diamidino-2-phenylindole (DAPI, A and B first panels). Co-localization of TIMP-1 and MMPs on the surface of PMNs was assessed using confocal microscopy (see merged images in the fourth panels in A and B). The white bars are 5 microns in length. In C-E, surface TIMP-1 or MMPs levels were quantified using image analysis software, as described in Methods. Data are mean + SEM; n = 4 air-exposed NHPs and n = 6 smoke-exposed NHPs (200–500 cells were analyzed per animal). In C-E, data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 compared with baseline or the group indicated.

Article Snippet: Full length murine Mmp-8 (residues 21–465) and murine Mmp-9 (residues 20–730) proteins and mutant murine Mmp-8 (residues 21–276) and murine Mmp-9 (residues 20–519) proteins containing the catalytic domains and hinge regions but lacking the COOH-terminal hemopexin domains were obtained from Abnova (Taipei City, Taiwan). (7-Methoxycoumarin-4-yl)-Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L2,3-diaminopropionyl)-Ala-Arg-NH 2 (McaPLGLDpaAR) was purchased from CalBiochem Novabiochem Corp. (San Diego, CA).

Techniques: Isolation, Confocal Microscopy, Software, Two Tailed Test