ABSTRACT
The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor alpha (TNFalpha) at the cell surface through the use of a specific inhibitor of the TNFalpha-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFalpha and makes it possible to assess, in the same measurement, the phenotype of TNFalpha(+)-secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFalpha+ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFalpha expression on the majority of CD3+ T cells and secretion of Th1-associated cytokines: interferon gamma, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14+ monocytes showed surface TNFalpha expression; in parallel, high amounts of soluble IL1beta, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viral-specific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFalpha+ monocytes, TNFalpha+/CD8+ T cells and TNFalpha+/CD8- T lymphocytes in association with an increased secretion of IFNgamma, IL6 and TNFalpha.
Subject(s)
Cytokines/biosynthesis , Flow Cytometry/methods , Leukocytes, Mononuclear/cytology , T-Lymphocyte Subsets/cytology , Tetradecanoylphorbol Acetate/analogs & derivatives , Adult , Flow Cytometry/instrumentation , Humans , Ionomycin/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Sensitivity and Specificity , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency.
