Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis
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Published on: March 2, 2011
B Ferry1, P Antrobus, I Huzicka
1Department of Immunology, The Churchill Hospital, Oxford Radcliffe Hospital, UK.
Insights
This study developed a rapid, cost-effective intracellular cytokine assay using whole blood. The assay accurately measures cytokine production in T cells, showing variations in healthy individuals and potential in atopic dermatitis patients.
Area of Science:
- Immunology
- Cellular Biology
- Clinical Diagnostics
Background:
- Cytokine characterization is crucial for understanding clinical conditions.
- Intracellular cytokine detection via flow cytometry offers single-cell resolution.
- A rapid, cost-effective assay using whole blood is needed for clinical application.
Purpose of the Study:
- To develop and validate an intracellular cytokine assay using unseparated whole blood.
- To assess cytokine expression (IL-2, IFN-gamma, IL-4) in T cell subsets.
- To evaluate the assay's reliability and applicability in clinical settings.
Main Methods:
- Intracellular cytokine staining on unseparated whole blood.
- Flow cytometry analysis of T lymphocyte subpopulations (CD4+, CD8+).
- Stimulation with phorbol 12-myristate acetate (PMA)/ionomycin.
Main Results:
- Optimal cytokine induction in CD8- T cells at 10h, and CD8+ T cells at 6-10h.
- Consistent cytokine levels observed over a 3-month period in healthy individuals.
- Significant differences in IFN-gamma and IL-4 expression in atopic dermatitis patients compared to controls.
Conclusions:
- The developed whole blood assay is reliable and reproducible for intracellular cytokine detection.
- The assay reveals significant heterogeneity in cytokine production among healthy individuals.
- This method shows promise for diagnosing and monitoring immune-related diseases like atopic dermatitis.
Abstract:
In 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- T cells, IL-2 and interferon-gamma (IFN-gamma) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8+ T cells IL-2 was optimally induced after 10 h and IFN-gamma after 6 h. The levels of IL-2 and IFN-gamma in CD8+ and CD8- 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+ (range 9.7-41.3) and CD3/IFN-gamma+ cells (10.1-44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3+/CD8+ peripheral blood T cells expressing IFN-gamma and an increased percentage of CD3+/CD8- T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.
