Effects of cell purification methods on CD11b and l-selectin expression as well as the adherence and activation of leucocytes

Macey, Marion G.; McCarthy, D. A.; Vordermeier, Susanne; Newland, Adrian C.; Brown, Kenneth A.

doi:10.1016/0022-1759(95)00003-s

Cited by 105 publications

(71 citation statements)

References 20 publications

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“…They possessed a high expression of HLA-DR, HLA-DQ, initiated an MLR and were very potent at presenting recall antigens at low target-to-effector ratio; properties that are characteristic of DC [26]. Isolating neutrophils and mononuclear leucocytes from blood often results in cell activation [27], and a similar caveat is likely to apply to the enrichment of circulating DC. Thus, the phenotype of DC described in our study may be more representative of activated DC than resting cells.…”

Section: Discussionmentioning

confidence: 99%

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

Brown

Bedford

Macey

et al. 1997

Clinical and Experimental Immunology

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SUMMARYTo investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0 . 001) and lymphocytes (mean 12%; P < 0 . 001). When the monolayers were pretreated with tumour necrosis factor-alpha (TNF-a) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0 . 001) and lymphocytes (mean 18%; P < 0 . 001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0 . 05), CD11c (P < 0 . 005) and CD54 (P < 0 . 005) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0 . 05) and the homing receptor CD62L (mean 14%; P < 0 . 001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the b 1 -integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0 . 05). Antibody blocking studies demonstrated that DC binding to untreated and TNF-a-treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti-CD18 and anti-CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0 . 001). Thus, the expression of both b 1 -and b 2 -integrins contributes to the adhesive interaction between DC and endothelium.

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Section: Discussionmentioning

confidence: 99%

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

Brown

Bedford

Macey

et al. 1997

Clinical and Experimental Immunology

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“…The mice were then mask-ventilated with 1.5% isoflurane and 0.8L/minute flow oxygen for maintenance anesthesia. Eight hundred to 900 μL of blood was withdrawn from the ascending vena cava with a 23-gauge needle/1 mL syringe containing 0.14 mL of undiluted citrate-phosphatedextrose (Sigma, Cat #C7165) (Leino, and Sorvajarvi, 1992;Repo, Jansson, and LeirisaloRepo, 1995;Macey, et al, 1995;Peter, et al, 1999).…”

Section: Blood Acquisitionmentioning

confidence: 99%

“…Erythrocyte lysing, sample fixation, and other sample preparation techniques used in flow cytometry are known to activate blood cells, particularly leukocytes, leading to an alteration in surface glycoprotein expression and intracellular processes (Macey, et al, 1995;Hageberg, and Lyberg, 2000;Alvarez, Toll, Rivas, and Estella, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…However, studies indicate that erythrocyte lysing reagents cause increased leukocyte expression of glycoprotein CD11b and shedding of granulocyte glycoprotein L-selectin (McCarthy, Macey, Cahill, and Newland, 1994;Macey, et al, 1995;Macey, et al, 1999;Alvarez-Larran, Toll, Rivas, and Estella, 2005). Erythrocyte lysing also causes other blood cells, including leukocytes and platelets, to lyse (Terstappen, Meiners, and Loken, 1989;Alvarez-Larran, et al, 2005), creating cell microparticles and debris that interact with and activate leukocytes (Repo, Jansson, and Leirisalo-Repo, 1993).…”

Section: Introductionmentioning

confidence: 99%

“…Because of this, Hageberg and Lyberg (2000), fixed samples before antibody staining and demonstrated a reduction in platelet-leukocyte conjugation, which they determined was closer to in vivo levels. Even though the use of sample fixation is controversial, it is often necessary, because cell interactions and surface antigen expression change over time (Macey, McCarthy, Vordermeier, Newland, and Brown, 1995;Hageberg, and Lyberg, 2000). Therefore, understanding the degree of cellular change caused by fixation during sample preparation and its effect on results is imperative to the development of flow cytometric methods with the least amount of experimental artifact possible.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

Maes

Davidson

McDonagh

et al. 2007

Journal of Immunological Methods

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Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra-and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 μg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5ug/ml PerCP-conjugated anti-CD45, 5ug/ml FITC-conjugated anti-CD11b, or 80 μM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior

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Quantifying rat pulmonary intravascular mononuclear phagocytes

Niehaus

Mehendale

1998

Anat. Rec.

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Cells of the mononuclear phagocyte system (MPS) protect the host by clearing effete and foreign particulates from the circulation. The current study was designed to identify, quantify, harvest, and provide a partial functional characterization of the systemic host-defense cell located in the pulmonary microvasculature of the rat. Critical colloid doses of test particulates (monastral blue B [MBB] or polystyrene beads) were infused intra-arterially into anesthetized rats so that phagocytically active pulmonary intravascular phagocytes could be identified. Morphologic characterization of in situ phagocytes was performed using electron microscopy. The number of active phagocytes was then determined using tissue samples processed for light microscopy. Finally, sequential perfusion of the pulmonary vasculature with buffer, chelating agent, and collagenase allowed elution and preliminary functional characterization of the pulmonary intravascular mononuclear phagocyte (PIMP). Electron microscopy demonstrated that both mononuclear phagocytes and neutrophils contributed to pulmonary sequestration of circulating particulates. Light microscopy showed that the microvasculature of each alveolus contained 0.50+/-0.19 active mononuclear phagocytes and 0.14+/-0.12 active neutrophils. A chelation/collagenase elution technique was then used to harvest the PIMP. Histologic evaluation of the postperfusion lungs indicated that 80% of the active phagocytes were removed by the technique. In total, the elution fluids contained 2.63+/-1.04 x 10(7) cells, with 1.60+/-0.78 x 10(7), 0.49+/-0.17 x 10(7), and 0.54+/-0.26 x 10(7) of those cells being mononuclear phagocytes, neutrophils, and lymphocytes, respectively. Functionally, the mononuclear phagocyte population exhibited a spectrum of phagocytic activities, with 51.5+/-19.5% of the cells being inactive, 33.9+/-13.4% exhibiting moderate phagocytic activity, and 14.6+/-9.8% demonstrating intensive phagocytic capacity. The current study provides the first quantified demonstration that mononuclear phagocytes are primarily responsible for sequestering blood-borne foreign particulates in the pulmonary circulation of the rat. Approximately 2 x 10(7) PIMP existed in the lungs of 300 gram rats. The functionally heterogeneous mononuclear phagocytes exhibited phagocytic capacities ranging from avidly phagocytic (14.6+/-9.8%) through moderately active (33.9+/-13.4%) to inactive. The lung microvasculature's large pool of inactive mononuclear phagocytes may provide a recruitable mechanism to allow significant increases in clearance of circulating particulates. A resident pool of activatable mononuclear phagocytes might explain previous clinical observations of increased particulate localization in the lung microvasculature of septic patients.

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Effects of cell purification methods on CD11b and l-selectin expression as well as the adherence and activation of leucocytes

Cited by 105 publications

References 20 publications

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

Human blood dendritic cells: binding to vascular endothelium and expression of adhesion molecules

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

Quantifying rat pulmonary intravascular mononuclear phagocytes

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