Purification of human blood eosinophils by negative selection using immunomagnetic beads

Hansel, Trevor T.; Pound, John D.; Pilling, Darrell; Kitas, George D.; Salmon, Mike; Gentle, T. A.; Lee, S.S.; Thompson, R. A.

doi:10.1016/0022-1759(89)90339-6

Cited by 184 publications

(84 citation statements)

References 10 publications

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“…Briefly, erythrocytes were removed by hypotonic lysis from the dextran sedimentation-isolated granulocyte fraction. The granulocytes were then incubated with anti-CD16 microbeads (Mitenyi Biotec Inc., Sunnyvale, CA) for 30 min (15) and were then passed through a MACS column (Mitenyi Biotec Inc.). The eosinophils collected in the flow-through were shown immunohistochemically to be greater than 99% pure.…”

Section: Methodsmentioning

confidence: 99%

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

Dairaghi¹,

Oldham

Bacon³

et al. 1997

Journal of Biological Chemistry

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The CC chemokine receptor 3 (CCR3) plays an important role in the regulation of the migration of eosinophils, a leukocyte population involved in many inflammatory pathologies including asthma. CCR3 binds to the CC chemokine eotaxin, a promigratory cytokine originally isolated as the key component in a model of eosinophil-induced airway inflammation. We show here that eotaxin/CCR3 binding interactions exhibit a marked sensitivity to relatively small changes in the extracellular environment. In particular, modest variations in the pH and the level of sodium chloride over a range of physiologic and near physiologic conditions had dramatic effects on eotaxin binding and CCR3-mediated cytoplasmic Ca 2؉ mobilization. These biochemical indices were reflected at the functional level as well; small changes in pH and salt also resulted in striking changes in the migration of primary human eosinophils in vitro. These results reveal that relatively small perturbations in extracellular buffer conditions can yield widely disparate interpretations of CCR3 ligand binding and affinities and suggest that modulation of the tissue microenvironment might be utilized to control the affinity and efficacy of chemokine-mediated cell migration.Eosinophils are involved in many inflammatory pathologies including asthma, urticaria, and hypereosinophilic syndrome (1-3). The CC chemokine receptor 3 (CCR3) 1 and its ligands, most notably eotaxin, have been shown to play a central role in controlling eosinophil migration (4 -12). Eotaxin was originally isolated as a protein responsible for eosinophil chemoattraction in the bronchoalveolar fluid of allergen-sensitized guinea pigs (9). Subsequently, the human and mouse homologs were identified and also shown to be chemoattractants for eosinophils (10,12). Eotaxin has been shown to be a primary ligand for the seven-transmembrane G protein-coupled receptor CCR3. The genes for CCR3 and the related receptors CCR1-CCR5 are encoded within a 130-kilobase region of human chromosome 3 (13) indicating recent gene duplication and divergence and perhaps explaining the partial overlap of chemokine ligand repertoires. CCR3 is highly expressed on primary human eosinophils (50,000 -400,000 sites/cell) (5-7), consistent with the potent ability of eotaxin to selectively influence the migration of these cells.The mechanisms by which specific cells are attracted by specific chemokines have been a topic of intense interest and clinical relevance. The question is complicated by the fact that a number of closely related chemokine receptors with overlapping specificities are expressed on many different classes of leukocytes. In addition, the affinity of a specific receptor/ligand interaction can vary depending upon the specific cell type expressing the chemokine receptor, perhaps due to posttranslational modification events or G protein coupling. Thus it appears that cells can modulate their responses to chemokines, but how this is done is not yet clear. While most investigations have focused on factors intrinsic to the...

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Section: Methodsmentioning

confidence: 99%

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

Dairaghi¹,

Oldham

Bacon³

et al. 1997

Journal of Biological Chemistry

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“…Eosinophils were purified from peripheral blood from nonallergic healthy donors (n ϭ 3), one patient with infectious eosinophilia (patient DA, 25% eosinophilic counts) and one CSS-patient (patient PF, 32% eosinophilic counts) using Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation and negative selection with an anti-CD16 monoclonal antibody and immunomagnetic beads coated with goat anti-mouse IgG (Miltenyi Biotec, Bergisch Gladbach, Germany) according to Hansel et al 18 The purity of eosinophils based on light microscopic examination of the purified cells after staining with DiffQuick (American Scientific Products, McGraw Park, IL) was Ͼ95% and viability was Ͼ98% as assessed by trypan blue (Sigma, Deisenhofen, Germany) exclusion. After purification, eosinophils were suspended in RPMI-1640 medium and supplemented with 10% fetal bovine serum (FBS).…”

Section: Purification Of Peripheral Blood Eosinophilsmentioning

confidence: 99%

Involvement of Soluble CD95 in Churg-Strauss Syndrome

Müschen

Warskulat

Perniok

et al. 1999

The American Journal of Pathology

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Deficiency of CD95 (Apo-1/Fas)-mediated apoptosis has recently been found in some autoimmune lymphoproliferative disorders due to inherited mutations of the CD95 gene. In this study , impairment of CD95 ligand-mediated killing of lymphocytes and eosinophils in Churg-Strauss Syndrome (CSS) , which was a result of variation of CD95 receptor isoform expression , is demonstrated. Compared to those from healthy individuals , peripheral blood lymphocytes from eight CSS patients exhibit a switch from the membrane-bound CD95 receptor expression to its soluble splice variant , which protects from CD95L-mediated apoptosis. In five out of seven CSS patients recurrent oligoclonal T cell expansions were found, all using a V␤-gene from the V␤21 family associated with similar CDR3 motifs , indicating the predominance of T cell clones of a similar specificity in the CSS patients. In two of them , the effect of immunosuppressive therapy was studied. In both cases aberrant overexpression of the soluble CD95 receptor isoform and deviations from normal TCR V␤-gene usage normalized in parallel with the clinical improvement. Furthermore , soluble CD95 was identified as a survival factor for eosinophils rescuing eosinophils from apoptosis in the absence of growth factors in vitro. Churg-Strauss Syndrome (CSS) was for the first time described by Lotte Strauss and Jacob Churg in 1951 in The American Journal of Pathology, 1 defining a new entity with systemic vasculitis, blood and tissue eosinophilia, and a long-term history of asthma. Systemic vasculitis in this rare syndrome mainly affects the lower respiratory tract, kidneys, skin, and nervous system. 2 The gastrointestinal tract is involved in about 35% of the CSS cases. 2 Infiltrating eosinophils are frequently found in granulomatous lesions 3 and the fraction of eosinophils in the peripheral blood correlates with the course of the disease, 3,4 suggestive of a role of eosinophils in the pathogenesis of CSS. 5 On the other hand, T cells were frequently implicated in autoreactivity and inflammatory lesions. 6 However, a potential role of T lymphocytes in CSS and the molecular mechanisms underlying this disease have not yet been studied.One tool to analyze T cell populations in clinical samples at the molecular level is the Immunoscope technique. 5 This approach is based on the hypothesis that the expression of unique, rearranged T cell receptor (TCR) genes reflects the specificity of a given T cell. 7 Each TCR-␤ chain consists of a variable (V), diversity (D), joining (J), and a constant region, the first three determining the antigen specificity in their VDJ-junction, including complementarity-determining region III (CDR3). During T cell differentiation, unique variable region genes are created by recombination of V, D and J segments for the TCR-␤ locus. More than 70 V␤ gene segments have been characterized and are classified into 24 gene families 8 ( Figure 1A).In T cells the CD95/CD95 ligand system is a major pathway of apoptotic cell death and thus essential for prevention of lymp...

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“…085 and 1 . 090 g/ml fraction of Percoll) containing neutrophils and eosinophils [16]; and second, extraction of the contaminating neutrophils using a MoAb (MG38; Nichirei, Tokyo, Japan) against CD16 (Fc°RIII) in a negative, indirect selection procedure using immunomagnetic beads (Advanced Magnetics, Cambridge, MA) [17]. Culture medium was RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; GIBCO), 2 mM Lglutamine.…”

Section: Isolation Of Human Blood Eosinophils and Culturementioning

confidence: 99%

IL-5 but not interferon-gamma (IFN-γ) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression

Ochiai

Kagami

Matsumura

et al. 1997

Clinical and Experimental Immunology

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Purification of human blood eosinophils by negative selection using immunomagnetic beads

Cited by 184 publications

References 10 publications

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

Involvement of Soluble CD95 in Churg-Strauss Syndrome

IL-5 but not interferon-gamma (IFN-γ) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression

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