Shape, F‐actin, and surface morphology changes during chemotactic peptide‐induced polarity in human neutrophils

Fernández-Segura, Eduardo; Peula-García, José Manuel; Santos, Juan Luis; Campos, Antonìo

doi:10.1002/ar.1092410410

Cited by 24 publications

(22 citation statements)

References 41 publications

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“…In contrast, activated cells with DMSO/fMLP (fmlp ϭ N-formylmethionyl-leveyl-phenylalanine) exposure had very irregular surfaces (Fernandez-Segura et al, 1995) as did resting monocytes (cf Fig. 1c) in the present study indicating that DMSO alone did not induce rounding.…”

Section: Discussioncontrasting

confidence: 77%

“…The DCF and DGO cells were rounder with a much smoother plasmalemmal surface similar to previous light and scanning electron microscope studies of unactivated neutrophils in suspension (Fernandez-Segura et al, 1995). In contrast, activated cells with DMSO/fMLP (fmlp ϭ N-formylmethionyl-leveyl-phenylalanine) exposure had very irregular surfaces (Fernandez-Segura et al, 1995) as did resting monocytes (cf Fig.…”

Section: Discussionsupporting

confidence: 52%

“…Volume fraction (V V ) was determined by the area fraction (A A ) based on the standard morphometric relation, V V ϭ A A , and reported as percent (Aherne and Dunnill, 1982). A cell form factor [FF C ϭ (4A)/P 2 ] with a value of 1.0 being a perfect circle (Fernandez-Segura et al, 1995) and the cell's surface/volume density [S VC ϭ (4/)(P/A)] (Weibel and Bolender, 1973) were calculated. Cell volume [V C ϭ (4/3) r 3 ] was calculated assuming the cell to be a sphere with radius based on profile area [r ϭ (3A/2)] (Sokol et al, 1987).…”

Section: Morphometric Analysismentioning

confidence: 99%

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Morphology of human neutrophils: A comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide

Gilbert

Parmley

1998

Anat. Rec.

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Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27-30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO.

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Section: Discussioncontrasting

confidence: 77%

Section: Discussionsupporting

confidence: 52%

Section: Morphometric Analysismentioning

confidence: 99%

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Morphology of human neutrophils: A comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide

Gilbert

Parmley

1998

Anat. Rec.

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“…Another phenomenon associated with neutrophil activation is an increase in cell size (14,17,22,31), which can be directly measured by flow cytometry as an increase in forward scatter. The forward scatter of infected cells increased directly with the bacterial load and duration of infection (Fig.…”

Section: Resultsmentioning

confidence: 99%

Anaplasma phagocytophilumUtilizes Multiple Host Evasion Mechanisms To Thwart NADPH Oxidase-Mediated Killing during Neutrophil Infection

et al. 2004

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Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O 2 ؊ ) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. ؊ in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22 phox and gp91 phox . Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.

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“…After washing, the leukocytes were resuspended in DPBS containing calcium and 0.5% bovine serum albumin (BSA; Sigma, St Louis, MO) to a concentration of 3 ϫ 10 6 cells/ml. To obtain "activated" neutrophils, the 10-ml blood specimens were immediately added to 0.1 ml 10 Ϫ 6 M N -formylmethionyl-leucyl-phenylalanine (FMLP; Sigma) in DPBS (Fernández-Segura et al 1995). After 1 min, the blood was added to 40 ml 0.25% paraformaldehyde in DPBS and the neutrophils isolated as described.…”

Section: Leukocyte Isolationmentioning

confidence: 99%

Receptors for the Chemoattractants C5a and IL-8 Are Clustered on the Surface of Human Neutrophils

Gray

Hasslen

Ember

et al. 1997

J Histochem Cytochem.

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SUMMARYWe have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N -formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

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Shape, F‐actin, and surface morphology changes during chemotactic peptide‐induced polarity in human neutrophils

Cited by 24 publications

References 41 publications

Morphology of human neutrophils: A comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide

Morphology of human neutrophils: A comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide

Anaplasma phagocytophilumUtilizes Multiple Host Evasion Mechanisms To Thwart NADPH Oxidase-Mediated Killing during Neutrophil Infection

Receptors for the Chemoattractants C5a and IL-8 Are Clustered on the Surface of Human Neutrophils

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