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

Abstract

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.

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

“…A contribution by several other cytokines, responsible for a proinflammatory response, such as IL-6 and TNF-␣, or responsible for an anti-inflammatory response, such as IL-10, has been suspected (11,12,21). Most studies have been performed on peripheral blood mononuclear cells (PBMC) and macrophages; only a few have been performed on whole blood (6,7). Using the FMBA we investigated the concentration of cytokines in whole blood of atopic and nonatopic asthmatics and in atopic and nonatopic controls.…”

Intracellular production of IL-2, IL-4, IFN-γ, and TNF-α by peripheral blood CD3+ and CD4+ T cells in children with atopic dermatitis

“…Whether this can influence subsequent clinical course of AD remains to be elucidated. Although we used flow cytometry to detect intracellular cytokine expression at the single-cell level, as with most authors of the above-cited reports, some differences in technique, including time stimulation, permeabilization, stimulation agents, Golgi apparatus blockers, antibody, and analyzed T cell subsets, could have an impact on our results [10].The small number of subjects may also be a drawback of this study; nevertheless, some differences among the particular subgroups were observed. In our study, we failed to demonstrate a role of IL-4 in patients with AD, but we showed significantly increased total IgE levels and numbers of circulating eosinophils in AD patients compared with the control group.…”

Fibrin network adaptation to cell-generated forces

“…Rheology is used to measure the viscoelastic properties of fibrin networks using different rheological techniques (Ferry et al 1997;Jansen et al 2013;Kurniawan et al 2016;Piechocka et al 2016). Ferry et al (1997) were the first to study the linear viscoelastic behavior of fibrin clots using torsional rheometry (Ferry et al 1997).…”