A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic Separation

Cotter, Matthew J.; Norman, Keith E.; Hellewell, Paul G.; Ridger, Victoria

doi:10.1016/s0002-9440(10)61719-1

Cited by 77 publications

(68 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cunnion et al demonstrated that mice depleted of complement by cobra venom factor were more susceptible to staphylococcal infection than control mice (7). Acapsular strains of S. aureus bind more C3 than encapsulated strains, although the exact mechanism for this is unclear (5,35). To investigate the influence of CP5 and CP8 production on complement deposition, we used flow cytometry to demonstrate the kinetics and amounts of C3b and iC3b deposition on Reynolds (CP5) and Reynolds (CP8).…”

Section: Discussionmentioning

confidence: 99%

Staphylococcus aureusStrains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence

Watts¹,

et al. 2005

View full text Add to dashboard Cite

Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5 ؉ strain showed a significantly higher bacteremia level than mice challenged with the CP8 ؉ strain. Similarly, the CP5 ؉ strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5 ؉ strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5 ؉ and CP8 ؉ strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.Staphylococcus aureus is a major bacterial pathogen that causes a wide spectrum of clinical infections, ranging from localized soft-tissue infections to life-threatening bacteremia and endocarditis (25). Many virulence factors contribute to the pathogenesis of staphylococcal infections, including surfaceassociated adhesins and secreted exoproteins and toxins (35). Like many invasive bacterial pathogens, S. aureus produces a capsular polysaccharide (CP) that enhances its resistance to clearance by host innate immune defenses. Most clinical isolates of S. aureus are encapsulated, and serotype 5 and 8 strains predominate (2,11,40). The type 5 (CP5) and type 8 (CP8) capsular polysaccharides have similar trisaccharide repeating units comprised of N-acetyl mannosaminuronic acid, N-acetyl L-fucosamine, and N-acetyl D-fucosamine (9, 28, 43). CP5 and CP8 are serologically distinct, and this can be attributed to differences in the linkages between the sugars and in the sites of O acetylation.Previous studies have correlated S. aureus capsule production with resistance to in vitro phagocytic uptake and killing (13, 41). Human neutrophils phagocytose capsule-negative mutants in the presence of nonimmune serum with complement activity, whereas serotype 5 isolates require both capsulespecific antibodies and complement for optimal opsonophagocytic killing (4, 41). Nilsson et al. (29) reported that peritoneal macrophages from mice phagocy...

show abstract

Section: Discussionmentioning

confidence: 99%

Staphylococcus aureusStrains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence

Watts¹,

et al. 2005

View full text Add to dashboard Cite

show abstract

“…All samples were resuspended in the wash buffer and subjected in full volume to flow cytometry analysis on a BD FACSCalibur flow cytometer (BD Biosciences). Buffy coats were obtained from mouse blood as previously described (51). All flow cytometry data were analyzed using FlowJo Mac, version 8.1.1 (Tree Star).…”

Section: Methodsmentioning

confidence: 99%

Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice

Zhang¹,

Clarke²,

Weiser³

2009

J. Clin. Invest.

332

440

View full text Add to dashboard Cite

Microbial colonization of mucosal surfaces may be an initial event in the progression to disease, and it is often a transient process. For the extracellular pathogen Streptococcus pneumoniae studied in a mouse model, nasopharyngeal carriage is eliminated over a period of weeks and requires cellular rather than humoral immunity. Here, we demonstrate that primary infection led to TLR2-dependent recruitment of monocyte/macrophages into the upper airway lumen, where they engulfed pneumococci. Pharmacologic depletion of luminal monocyte/macrophages by intranasal instillation of liposomal clodronate diminished pneumococcal clearance. Efficient clearance of colonization required TLR2 signaling to generate a population of pneumococcal-specific IL-17-expressing CD4 + T cells. Depletion of either IL-17A or CD4 + T cells was sufficient to block the recruitment of monocyte/macrophages that allowed for effective late pneumococcal clearance. In contrast with naive mice, previously colonized mice showed enhanced early clearance that correlated with a more robust influx of luminal neutrophils. As for primary colonization, these cellular responses required Th17 immunity. Our findings demonstrate that monocyte/macrophages and neutrophils recruited to the mucosal surface are key effectors in clearing primary and secondary bacterial colonization, respectively.

show abstract

“…Ethical approval was obtained from the South Sheffield Research Ethics Committee, and all participants gave written informed consent in accordance with the Declaration of Helsinki principles. Murine bone marrow-derived neutrophils were isolated using modified discontinuous HBSS-Percoll gradients (82%, 62%, and 51%) and peripheral blood neutrophils by negative magnetic selection following a terminal inferior vena cava bleed into a 23 gauge pre-heparinized needle (42). Cell purity, assessed by cytospin, was routinely greater than 97% human peripheral blood, greater than 70% murine bone marrow, and greater than 80% murine peripheral blood.…”

Section: Methodsmentioning

confidence: 99%

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice

Walmsley¹,

Chilvers²,

Thompson³

et al. 2011

J. Clin. Invest.

164

176

View full text Add to dashboard Cite

The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-x L . In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.

show abstract

A Novel Method for Isolation of Neutrophils from Murine Blood Using Negative Immunomagnetic Separation

Cited by 77 publications

References 64 publications

Staphylococcus aureusStrains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence

Staphylococcus aureusStrains That Express Serotype 5 or Serotype 8 Capsular Polysaccharides Differ in Virulence

Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice

Contact Info

Product

Resources

About