1. Siglec-E is a negative regulator of acute pulmonary neutrophil inflammation and suppresses CD11b β2-integrin–dependent signaling
by Sarah J. McMillan, Ritu S. Sharma, Emma J. McKenzie, Hannah E. Richards, Jiquan Zhang, Alan Prescott, and Paul R. Crocker
Blood
Volume 121(11):
March 14, 2013
©2013 by American Society of Hematology

2. Generation of siglec-E–deficient mice.
Generation of siglec-E–deficient mice. (A) Schematic representation of Siglec-E locus, the gene targeting vector, and the predicted mutated Siglec-E gene. (B) Targeting vector to introduce R126D mutation in exon 1 (that contains the sialic acid binding site), with the aim of generating a full-length protein that lacks the ability to bind sialic acid. (C) Lysates from bone marrow neutrophils or bone marrow–derived macrophages cultured for 3 days in the presence of 100 ng/mL LPS to induce siglec-E were immunoprecipitated with sheep anti-mouse siglec-E IgG and subsequently immunoblotted with sheep anti-mouse siglec-E antiserum followed by rabbit anti-sheep IgG-HRP conjugate. *An upper nonspecific band and a lower heavy chain band were common to all samples.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

3. LPS-induced airway inflammation in siglec-E–deficient mice.
LPS-induced airway inflammation in siglec-E–deficient mice. Total and differential cell counts in (A) bronchoalveolar lavage and (B) lung tissue digests from WT mice and siglec-ER126D mice following aerosolized LPS. Values are expressed as means ± SEM; n = 4-6 per group; and *P < .05, Mann-Whitney U test compared with WT mice. (C) Paraffin-embedded lung tissue sections were stained with a specific antibody against myeloperoxidase to identify neutrophils (arrows). Scale bars represent 20 µm on images acquired with an Axioskop Zeiss microscope (63× PlanNeoFluor/1.25 objective) equipped with an Axiocam digital camera (Carl Zeiss Microscopy LLC, Thornwood, New York). Data are representative of n = 4-6 per group. (D) Eight weeks after reconstitution, radiation bone marrow chimeric mice were exposed to aerosolized LPS. One hour after LPS, cells were isolated from lung tissue digests, and numbers of neutrophils were determined by flow cytometry. Data are expressed as means ± SEM; n = 4-5 per group; and *P < .05, Mann-Whitney U test compared with mice reconstituted with WT cells. (E) Increased margination of neutrophils in the lung tissue of siglec-E–deficient mice 4 hours after 50 μg intraperitoneal LPS. Paraffin-embedded lung tissue sections were stained with a specific antibody against myeloperoxidase to identify neutrophils in the capillary bed (arrows; scale bars represent 20 µm). Neutrophils were enumerated in 10 random fields per section. Values are expressed as means ± SEM; n = 5 per group; and *P < .05, Mann-Whitney U test compared with WT mice.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

4. Chemotactic responses and requirement for CD11b in exaggerated neutrophil recruitment of siglec-E–deficient mice exposed to LPS. (A) CXCL1 and CXCL2 levels were measured in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue homogenate by enzyme-link...
Chemotactic responses and requirement for CD11b in exaggerated neutrophil recruitment of siglec-E–deficient mice exposed to LPS. (A) CXCL1 and CXCL2 levels were measured in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue homogenate by enzyme-linked immunosorbent assay from WT mice and siglec-ER126D mice 30 minutes after LPS aerosol. Data are expressed as means ± SEM; n = 4-5 per group. (B) A 1:1 mix of WT and siglec-E−/− mice bone marrow cells were placed into transwell chemotaxis chambers, and the number of cells migrating was determined by flow cytometry. Data are expressed as means ± SEM of triplicate wells. (C) Mice were injected intravenously with 100 µg anti-CD11b antibody (5C6 clone 1) or rat IgG immediately preceding exposure to aerosolized LPS and cellular recruitment analyzed at 3 hours in BAL or in lung tissue digests (Lung). Data are expressed as means ± SEM; n = 4-9 per group from 2 independent experiments; and *P < .05, Mann-Whitney U test compared with WT mice.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

5. Siglec-E does not regulate “inside-out” signaling of CD11b in neutrophils.
Siglec-E does not regulate “inside-out” signaling of CD11b in neutrophils. (A) Whole blood or bone marrow cells from WT and siglec-E−/− mice, were incubated for 30 minutes at 37°C with the indicated stimuli. Surface expression of CD11b and CD62L on mature neutrophils (SSCintGr-1hiCD11b+) was assessed by flow cytometry. Data are expressed as mean ± standard deviation; n = 3 for blood and for bone marrow as the mean, representative of 2 independent experiments. (B) Adhesion of WT and siglec-E−/− bone marrow cells to fibrinogen (Fb) in the presence and absence of PDBu. To investigate integrin-dependent adhesion, cells were pretreated with 20 μg/mL anti-CD11b antibody (5C6 clone 1) or 10 mM EDTA 20 minutes before plating. Data are expressed as mean ± SEM with 5 replicates for each data point and are representative of 2 independent experiments.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

6. Siglec-E is preferentially localized in areas of high CD11b staining on neutrophils spreading over fibrinogen.
Siglec-E is preferentially localized in areas of high CD11b staining on neutrophils spreading over fibrinogen. WT and siglec-E−/− bone marrow cells were plated onto fibrinogen-coated coverslips for 20 minutes at 37°C. Neutrophils were fixed with 3.7% formaldehyde in phosphate-buffered saline and stained with polyclonal anti–siglec-E antibody conjugated to FluoProbe 547H and/or anti–CD11b-Alexa488 antibody. The cells were mounted in the presence of 4,6 diamidino-2-phenylindole to distinguish neutrophil nuclear morphology and were analyzed by confocal microscopy (LSM 700 microscope and Zen 2009 software; Carl Zeiss). Images were collected with an α-Plan-Apochromat × 100 NA 1.46 objective. Scale bars represent 5 µm. Image analysis was performed using Volocity software. The number of siglec-E foci per cell was quantified in areas of high CD11b staining compared with areas of low CD11b staining. High CD11b intensity was set to 1 to 3.3 standard deviations from the mean intensity, and low CD11b intensity was set to 0 to 1 standard deviations. The number of siglec-E foci was measured automatically in each region. Data are expressed as mean ± SEM from 10 images from 2 independent experiments containing ≥4 cells per image; *P < .05, paired t test.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

7. Siglec-E is a negative regulator of CD11b-dependent phosphorylation of Syk and p38 MAP kinase.
Siglec-E is a negative regulator of CD11b-dependent phosphorylation of Syk and p38 MAP kinase. (A) Increased phosphorylation of Syk and p38 MAP kinase in siglec-E−/− cells following adhesion to fibrinogen. WT and siglec-E−/− bone marrow cells were plated at 37°C under the different conditions and times indicated, and whole cell lysates were immunoblotted with antibodies against phopho-Y317Syk, total Syk, phospho-p38, and total p38. As a negative control, cells were treated with the Src family kinase inhibitor PP2 (20 µM). As a positive control, cells were treated with freshly prepared pervanadate (PV) to inhibit cellular tyrosine phosphatases. Phospho-Syk and phospho-p38 signals for each sample were normalized to their respective total Syk or total p38 signals. Data are representative of 3 independent experiments. Bar charts show densitometry expressed as means ± SEM; n = 3 per group; and *P < .05, paired t test compared with WT control. (B) Siglec-E binds fibrinogen in a sialic acid–dependent manner. Siglec-E-Fc precomplexes were incubated on fibrinogen pretreated with or without sialidase. Bar charts show densitometry expressed as means ± SEM; n = 3 per group; and *P < .05, paired t test compared with untreated membranes. (C) Sialidase treatment of fibrinogen reverses siglec-E–dependent inhibition of phopho-Y317Syk. Wells were coated with fibrinogen or IgG/anti-IgG complexes and either left untreated (No buffer) or were treated with V. cholerae sialidase (Sialidase) or sialidase buffer alone (Buffer) for 60 minutes. After blocking with 10% fetal calf serum, bone marrow cells were allowed to adhere for 20 minutes. Lysates were immunoblotted with antibodies to phospho-Y317Syk antibody and total Syk. Bar charts show densitometry expressed as means ± SEM; n = 4 per group; and *P < .05, paired t test compared with WT control.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology

8. Siglec-E associates with SHP-1 in the absence of tyrosine phosphorylation.
Siglec-E associates with SHP-1 in the absence of tyrosine phosphorylation. WT and siglec-E−/− bone marrow cells were plated on fibrinogen or stimulated in suspension with either pervanadate (PV) or with LPS (10 µg/mL) for the indicated time periods at 37°C. Lysates were immunoprecipitated with anti–siglec-E antibody followed by immunoblotting with anti–siglec-E, anti–SHP-1, anti–SHP-2, and anti-pY antibodies. As a positive control for siglec-E and SHP-2, lysates from WT and siglec-E–transfected CHO cells were analyzed in parallel. Data are representative of 3 independent experiments.
Sarah J. McMillan et al. Blood 2013;121:
©2013 by American Society of Hematology