Detecting cytokine production at the single-cell level

Lewis, Claire E.

doi:10.1016/1043-4666(91)90014-5

Cited by 17 publications

(8 citation statements)

References 30 publications

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“…Erythroid progenitors, which are known to be target of a number of growth factors (Metcalf 1984;Whetton and Dexter 1989), are rarely demonstrated to produce any regulatory molecules by themselves (Sytkowski et al 1990;Hermine et al 1991). This might be due to the fact that methods measuring bulk of cytokine release in culture supernatants, are not as sensitive as the RHPA (Lewis et al 1989;Lewis 1991) which allows cellular products of less than 10 is M to be discerned at the sin Bars also indicate the median of plaque sizes formed by the total of secretory cells as well as by monocytes/macrophages (mo/ma), neutrophil granulopoietic (gran) and erythroid (eryth) cells gle cell level. Moreover, cytokine production by erythroid cells possibly is a transient event that may be masked by predominant secretory activity of other marrow cell types under test conditions commonly applied.…”

Section: Discussionmentioning

confidence: 99%

Cytokine release by human bone marrow cells: analysis at the single cell level

Rohde

Wickenhauser

Denecke

et al. 1994

Vichows Archiv A Pathol Anat

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Regulation of haemopoiesis is closely mediated by a number of growth factors in the marrow microenvironment. The identification of the cell type secreting these regulatory polypeptides is difficult due to the heterogeneity of bone marrow cells. To analyse the release of haemopoietic growth factors by normal human bone marrow cells at the single cell level, we employed the reverse haemolytic plaque assay (RHPA). Freshly isolated human marrow cells were examined for the release of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6 and granulocyte-monocyte colony stimulating factor (GM-CSF). In order to identify various cytokine-secreting cell types, the RHPA was combined with immunocytochemical or enzymatic staining. The total of secreting marrow cells as well as the amount of several secretory haemopoietic subpopulations could be determined with this technique under various conditions. Following incubation with pure serum-free medium without addition of any mediator, only few cells secreting either IL-1 alpha, IL-3, IL-6 or GM-CSF could be observed. After 2 h incubation with recombinant human-IL-1 alpha (rhIL-1 alpha) (10.0 ng/ml) or rhGM-CSF (10.0 pg/ml) the number of cytokine-secreting cells significantly increased for all secretory products tested. Using cytochemical staining reactions, we were able to identify 55% of all cells secreting a specific cytokine. Glycophorin C-positive erythropoietic cells turned out to be the largest fraction (up to 89%) of cytokine-releasing haemopoietic cells, followed by neutrophil granulocytes (between 6 and 48%), and monocytes/macrophages (between 4 and 23%). Only few CD 61-positive cytokine-secreting megakaryocytes could be detected. Dose- and time-dependent kinetics after stimulation with rhGM-CSF revealed that the bulk of secretory activity originates from haemopoietic or rather from erythropoietic cells following low level stimulation and after short stimulation time. Thus, our data are in keeping with the assumption, that especially erythropoietic cells are producing a repertoire of cytokines that is thought to exhibit regulatory functions within marrow microenvironment. In the present study the RHPA is presented as an appropriate tool for measuring cytokine release not only of cells of the haematopoietic system but also of other tissues, for example solid tumours or malignant lymphomas.

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Section: Discussionmentioning

confidence: 99%

Cytokine release by human bone marrow cells: analysis at the single cell level

Rohde

Wickenhauser

Denecke

et al. 1994

Vichows Archiv A Pathol Anat

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“…It is clear, however, that cytokine production at the population level may not reflect expression at the single-cell level [10,11]. Assays used to examine cytokine production in single cells (reviewed in [12]) include ELISPOT, immunofluorescence microscopy [13], limiting dilution analysis [14] and in situ hybridization [10]. While informative, all these techniques are labour intensive or technically demanding, and examination of individual cells is severely limited in number.…”

Section: Introductionmentioning

confidence: 99%

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

Ferry

Antrobus

Hužička

et al. 1997

Clinical and Experimental Immunology

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SUMMARYIn recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8 ¹ T cells, IL-2 and interferon-gamma (IFN-g) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8 þ T cells IL-2 was optimally induced after 10 h and IFN-g after 6 h. The levels of IL-2 and IFN-g in CD8 þ and CD8 ¹ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2 þ (range 9 . 7-41 . 3) and CD3/IFN-g þ cells (10 . 1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n ¼ 5) there was a significantly decreased percentage of CD3 þ /CD8 þ peripheral blood T cells expressing IFN-g and an increased percentage of CD3 þ /CD8 ¹ T cells expressing IL-4 compared with non-atopic dermatitis controls (n ¼ 5). Possible applications of this technique are discussed.

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“…Several techniques have been used to analyze cytokine synthesis ex vivo at the single-cell level: ELISPOT, in situ hybridization, limiting dilution, and single-cell PCR (29). However, all of these techniques have significant drawbacks, requiring either high technical proficiency or laborious manual scoring of data.…”

Section: Introductionmentioning

confidence: 99%