A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies

Lichtenberger, Cornelia; Zakeri, Schaker M.; Baier, K.; Willheim, Martin; Holub, Margareta; Reinisch, Walter

doi:10.1016/s0022-1759(99)00076-9

Cited by 22 publications

(20 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To generate a preparation of highly purified neutrophils, cells isolated on a Ficoll-Paque density gradient were simultaneously incubated with anti-CD3, anti-CD19, and anti-HLA-DR antibodies conjugated to FITC, as well as a PE-conjugated anti-CD16 antibody. Dual label fluorescence-activated cell sorting (FACS) was then carried out to separate CD16 positive cells from cells that express CD3, CD19, or HLA-DR (66). With this protocol, a homogeneous population of cells (Ͼ99.9% neutrophils, Fig.…”

Section: Resultsmentioning

confidence: 99%

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

Dégousée¹,

Ghomashchi²,

Stefanski³

et al. 2002

Journal of Biological Chemistry

Self Cite

165

View full text Add to dashboard Cite

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A 2 (PLA 2 ) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA 2 (GV and GX sPLA 2 ) mRNA and contain GV and GX sPLA 2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA 2 s are undetectable. GV sPLA 2 is a component of both azurophilic and specific granules, whereas GX sPLA 2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA 2 and most, if not all, of the PLA 2 activity in the extracellular fluid of fMLPstimulated neutrophils is due to GV sPLA 2 . GV sPLA 2 does not contribute to fMLP-stimulated leukotriene B 4 production but may support the anti-bacterial properties of the neutrophil, because 10 -100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA 2 -␣ (group IV PLA 2 ␣), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B 4 in fMLP-and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

show abstract

Section: Resultsmentioning

confidence: 99%

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

Dégousée¹,

Ghomashchi²,

Stefanski³

et al. 2002

Journal of Biological Chemistry

Self Cite

165

View full text Add to dashboard Cite

show abstract

“…Most published observations on this subject were made in isolated neutrophils purified by Percoll or Ficoll separation. Although a purity of 98 % can be achieved by these methods, the few percent of monocytes left are sufficiently potent to release the low amounts of cytokines measured in such experiments (Kurt-jones et al, 2002;Lichtenberger et al, 1999;Reglier et al, 1998). Measurement of cytokines locked inside the cells inhibited in their Golgi function (by Brefeldin A) is a useful method of pinpointing the cells which produce the cytokine in response to a stimulus, as these can be clearly identified by counterstaining.…”

Section: Discussionmentioning

confidence: 99%

Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment

et al. 2003

View full text Add to dashboard Cite

Lipoteichoic acid (LTA) is a major immunostimulatory principle of Gram-positive bacteria. Intranasal application of LTA from S. aureus to mice resulted in greatly increased neutrophil and macrophage counts in the bronchoalveolar lavage as well as increased levels of the chemokine KC. The potential of highly pure, bioactive LTA from S. aureus to induce neutrophil recruitment and activation was investigated further in the human system. Although neutrophils expressed the key known receptors, CD14, TLR2 and TLR6, LTA did not induce or prime neutrophils for oxidative burst, or release of chemokines, bactericidal permeability-increasing protein or myeloperoxidase. However, LTA induced a strong release of the chemoattractants LTB 4 , IL-8, C5a, MCP-l and the colony-stimulating factor G-CSF in whole blood comparable to stimulation with the same concentration of LPS (S. abortus equi). Further, the cytokine and chemoattractant pattern induced by LTA correlated well with that induced by live S. aureus of the same strain. LTA does not appear to activate neutrophils directly, but is a strong stimulus for the recruitment of phagocytes to the site of infection.Abbreviations: BALF = bronchoalveolar lavage fluid; BPI = bacterial permeability-increasing protein; C5a = complement factor 5a; fMLP = N-formyl-L-methionyl-L-leucyl-Lphenylalanine; G-CSF = granulocyte colony-stimulating factor; LTA = lipoteichoic acid; LT = leukotriene; MCP-l = macrophage chemoattractant protein-l; MPO = myeloperoxidase; PMA = phorbol myristate acid; TLR = toll-like receptor; TNF = tumor necrosis factor.

show abstract

“…Antigen-containing neutrophils were found in lymphatic vessels and paracortical areas of the lymph nodes. Finally, major histocompatibility complex class II expression on human neutrophils has been demonstrated, and it is possible that neutrophils act as antigen-presenting cells themselves (13,16,20).…”

Section: Discussionmentioning

confidence: 99%

Neutrophil Depletion duringToxoplasma gondiiInfection Leads to Impaired Immunity and Lethal Systemic Pathology

et al. 2001

View full text Add to dashboard Cite

The immunomodulatory role of neutrophils during infection with Toxoplasma gondii was investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4 ؉ T cells, CD8 ؉ T cells, and NK1.1 ؉ cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.The immune system is a complex nonlinear system, involving the coordination of multiple cell types. Disease often results from an insufficient immune response. Lack of protection can occur when any one or more of the components of the immune system are impaired. Neutropenia is a risk factor associated with Aspergillus fumigatus, Candida albicans, Strongyloides ratti, Yersinia enterocolitica, Chlamydia trachomatis, Mycobacterium tuberculosis, Listeria monocytogenes, and Toxoplasma gondii infections (2,3,5,7,8,22,27,31,41). In some cases, neutropenia has been correlated with impaired protective acquired immunity, suggesting that neutrophils function as immunomodulators of acquired immunity (27, 31). The list of microbes affected by the actions of neutrophils is ever-growing, and it is evident that these cells are involved in the immune response to a highly diverse array of both intracellular and extracellular pathogens.We examined the development of immunity during T. gondii infection in mice depleted of neutrophils by monoclonal antibody (MAb) administration. T. gondii is an obligate intracellular protozoan parasite that poses an important public health risk. Congenital infections may result in severe birth defects, and reactivation of chronic infection can lead to development of encephalitis, a particular problem for persons who are immunosuppressed (25, 28). We found that neutrophil depletion at the time of infection led to development of lesions in multiple organs, including the spleen, lung, liver, and brain, and was associated with an impaired ability to produce early gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣) and interleukin-12 (IL-12). Splenic populations of T cells and NK cells were decreased in neutrophil-depleted infected mice. Moreover, neutrophil-depleted mice harbored an increased parasite burden. We conclude that neutrophils are important immunomodulators early in the course of T. gondii infection and play a critical role in protecting the host from uncontrolled tachyzoite replication. MATERIALS AND METHODSMice. C57BL/6 female mice (6 to 12 weeks of age) were obtained fro...

show abstract

A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies

Cited by 22 publications

References 25 publications

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

Groups IV, V, and X Phospholipases A2s in Human Neutrophils

Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment

Neutrophil Depletion duringToxoplasma gondiiInfection Leads to Impaired Immunity and Lethal Systemic Pathology

Contact Info

Product

Resources

About