1. Neutrophil elastase depends on serglycin proteoglycan for localization in granules
by Carsten U. Niemann, Magnus Åbrink, Gunnar Pejler, Rikke L. Fischer, Erik I. Christensen, Stefan D. Knight, and Niels Borregaard
Blood
Volume 109(10):
May 15, 2007
©2007 by American Society of Hematology

2. Characterization of bone marrow cells from wild-type (+/+), heterozygous (+/−), and serglycin knockout (−/−) mice.
Characterization of bone marrow cells from wild-type (+/+), heterozygous (+/−), and serglycin knockout (−/−) mice. (A-B) Giemsa staining of bone marrow cells from wild-type (A) and serglycin−/− mice (B). Murine neutrophils are characterized by donut-shaped nuclei with increasing lobulation during final maturation; bar, 20 μm. (C-D) Ultrastructure by electron microscopy of neutrophils from wild-type (C) and serglycin−/− mice (D). Higher magnifications of areas with granules from wild-type (E) and serglycin−/− neutrophils (F) show the same electron density of granules from the 2 genotypes. (G) Northern blotting with full-length serglycin probe. (H) Ethidium bromide staining of the gel before blotting, for control of equal loading; 5 μg total RNA from bone marrow cells per lane.
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Immunostaining for serglycin in bone marrow cells from wild-type and serglycin knockout mice and detection of 35S-labeled macromolecules.
Immunostaining for serglycin in bone marrow cells from wild-type and serglycin knockout mice and detection of 35S-labeled macromolecules. (A-B) Immunocytochemistry for serglycin; bar, 20 μm; neutrophils and precursor cells from wild-type mouse (A) and serglycin knockout mouse (B). (C) Western blotting of 1 ng recombinant murine serglycin per lane; detection with rabbit anti–murine serglycin IgG, blocked with 0 to 100 ng/mL murine serglycin as indicated above each lane. As seen, the immunoreactivity of the antibody is blocked in a dose-dependent manner by addition of the antigen in solution to the antibody. (D) Immunoelectron microscopy showing serglycin immunoreactivity (10 nm gold particles) in a myeloid cell from wild-type bone marrow; Golgi stacks are indicated by arrows. (E-F) Biosynthesis of 35S-labeled macromolecules in bone marrow cells and SDS-PAGE of cell lysates with (+) or without (−) cABC treatment. (E) Murine bone marrow cells from wild-type (+/+) and serglycin knockout mice (−/−). (F) Myeloid cells from human bone marrow and peripheral blood separated according to maturational stage; the designation of cell populations indicates the major cell types in each group (PMN, peripheral blood neutrophils; BC+SC, band cells and segmented neutrophils; MC+MM, myelocytes and metamyelocytes; MB+PM, myeloblasts and promyelocytes).
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

4. Neutrophil elastase in bone marrow cells from wild-type (+/+), heterozygous (+/−), and serglycin knockout (−/−) mice.
Neutrophil elastase in bone marrow cells from wild-type (+/+), heterozygous (+/−), and serglycin knockout (−/−) mice. (A) Elastase activity measured by spectrophotometry following the degradation rate for methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide. The SLPI-inhibitable activity is expressed as absorbance change per minute at 405 nm. Median is indicated by bold line and range by whiskers. The asterisk signifies statistically significant difference at the 5% level, log10-transformed data, independent t test; +/+, n = 3; +/−, n = 4; −/−, n = 6. (B) Western blotting of bone marrow cell lysates. The 2 blots have been loaded and processed in parallel; the lower blot has been incubated with rabbit anti–murine neutrophil elastase (NE), and the upper blot has been incubated with monoclonal mouse anti–murine β-actin (Ac) for loading control. Molecular weight markers are indicated. (C) Northern blotting with a full-length probe for murine neutrophil elastase; the blot is the same as in Figure 1 after stripping and reprobing with the neutrophil elastase probe. (D-E) Immunocytochemistry for neutrophil elastase in murine bone marrow cells from wild-type mice (D) and serglycin−/− mice (E); bar, 20 μm. Rarely a promyelocyte from serglycin−/− mice stains positive for neutrophil elastase (E, inset).
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

5. Immunocytochemistry and Western blotting for granule proteins.
Immunocytochemistry and Western blotting for granule proteins. (A-J) Immunocytochemistry for neutrophil granule proteins; bar, 20 μm; arrows point toward promyelocytes and arrowheads toward myelocytes. (A,C,E,G,I) Bone marrow cells from wild-type mice. (B,D,F,H,J) Bone marrow cells from serglycin knockout mice. (A-B) MPO and (C-D) BPI protein, azurophil granule proteins, (E-F) 24p3 and (G-H) lactoferrin (specific granule proteins), and (I-J) gelatinase B (MMP-9) (gelatinase granule protein). (K-N) Western blotting for 24p3 (K), lactoferrin (L), MMP-9 (M), and MPO (N), lysates of bone marrow cells from wild-type (+/+) and serglycin knockout mice (−/−); molecular weight markers are indicated.
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

6. The serine proteases cathepsin G and proteinase 3 in wild-type (+/+) and serglycin knockout mice (−/−) bone marrow cells.
The serine proteases cathepsin G and proteinase 3 in wild-type (+/+) and serglycin knockout mice (−/−) bone marrow cells. (A) Proteinase 3 activity (+/+, n = 3; −/−, n = 6) was measured spectrophotometrically by the degradation rate for methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide. The activity expressed as absorbance change per minute at 405 nm is the activity not inhibitable by SLPI. (B) Cathepsin G activity (+/+, n = 3; −/−, n = 3) was measured spectrophotometrically by dissociation rate for N-succinyl-Ala-Ala-Pro-Phe-P-nitroanilide at 410 nm. No statistically significant differences (5% level, log10-transformed data, independent t test) in serine protease activities could be detected between wild-type and serglycin−/− bone marrow cells. Median is indicated by bold line and range by whiskers. (C) Western blotting for cathepsin G (CG); MPO is used as an internal loading control. The 2 blots represent the upper and lower part of the same blot cut into 2 before antibody incubation. Molecular weight markers are indicated. (D-E) Immunocytochemistry for cathepsin G on bone marrow cells from wild-type (D) and serglycin−/− mice (E); bar, 20 μm.
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

7. Three-dimensional molecular models of neutrophil elastase, proteinase 3, and cathepsin G. The basis for modeling is described in “Materials and methods.” Residues with positively charged side chains are indicated by blue; those with negatively charged side ...
Three-dimensional molecular models of neutrophil elastase, proteinase 3, and cathepsin G. The basis for modeling is described in “Materials and methods.” Residues with positively charged side chains are indicated by blue; those with negatively charged side chains are indicated by red. Neutrophil elastase and cathepsin G are modeled with an inhibitor in the active site (orange). The putative glycosaminoglycan-binding regions around the active sites of the enzymes are encircled (yellow). (A-D) Murine neutrophil elastase, (E-F) murine cathepsin G, (I-L) murine proteinase 3. For each protein model, 4 different views separated by sequential 90-degree clockwise rotations around the vertical axis are shown.
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology

8. In vivo virulence of K pneumoniae in a peritonitis/sepsis model in wild-type (+/+) and serglycin knockout (−/−) mice.
In vivo virulence of K pneumoniae in a peritonitis/sepsis model in wild-type (+/+) and serglycin knockout (−/−) mice. Mice were challenged with K pneumoniae intraperitoneally at 0 hours. Six animals from each genotype were killed at 1 hour and 5 hours after bacterial injection, and CFU counts were determined for blood and peritoneal fluid (perit) samples for each time point (1 and 5). Gray line shows the median; ♦, single experiments; *, statistically significant difference at the 5% level, Mann-Whitney nonparametric test.
Carsten U. Niemann et al. Blood 2007;109:
©2007 by American Society of Hematology