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Expression and kinetics of cytokines determined by intracellular staining using flow cytometry

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Abstract

Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon γ (IFNγ) and tumor necrosis factor α (TNFα) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNγ and TNFα in lymphocyte subpopulations showed that TNFα was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNγ production was at 8 h, and production of IL2 exceeded that of IFNγ in T-cells at all times. IL2 production declined markedly after 8 h, while IFNγ remained relatively stable for 24 h. IL2 and TNFα were mainly produced by Th cells while CTL primarily expressed IFNγ. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.

Introduction

Intracellular staining of cytokines using flow cytometry is still a relatively novel method that allows simultaneous staining of cytokines and surface markers. It permits an identification of subpopulations and their cytokine production without prior cell sorting or the use of clones (Jung et al., 1993; Vikingsson et al., 1994; Prussin and Metcalfe, 1995).

Lymphocytes can be subdivided into naive and memory cells, the latter having been primed by an antigenic stimulus (Sanders et al., 1988). These subpopulations can be identified by their expression of variant CD45 isoforms that are formed from a single gene by differential splicing. Naive cells carry the CD45RA isoform while memory cells express the CD45R0 epitope (Vitetta et al., 1991). Memory cells are expected to mount a secondary immune response to a previously encountered antigen faster and more effectively than non-primed lymphocytes. While naive cells are believed to produce mainly IL2, fully developed Th1 and Th2 cells are thought to express the whole range of cytokines which is characteristic for the memory phenotype (Swain et al., 1996). We were therefore interested in determining whether naive and memory cells display different cytokine profiles and if the kinetics of cytokine expression differs between these subpopulations.

T-cells as the main producers of cytokines are classically subdivided into cytotoxic (CD3+CD8+) and T helper cells (CD3+CD4+ or CD3+CD8−). Using T-cell clones, cytokine production could be shown to be different in these two subsets; Th cells produced a wide range of cytokines such as IL2, IL4 and IFNγ while cytotoxic T-cells produced primarily IFNγ (Salgame et al., 1991). Using intracellular staining for flow cytometry we wished to evaluate cytokine profiles in these subsets and to determine whether these profiles changed over time. Moreover, we were interested to evaluate whether TNFα, which is often classified as a monokine, is also expressed in lymphocytes.

Using several controls we were able to show that intracellular staining of cytokines using flow cytometry provides a reliable method for the detection of cytokines at the single cell level. Our results may provide further insights into the kinetics and expression of cytokines in lymphocyte subpopulations.

Section snippets

Antibodies and reagents

Antibodies to the surface epitopes CD3 (clone UCHT1), CD8 (RPA-T8), CD45RA (HI100) and CD45R0 (UCHL1) as well as the anti-cytokine antibodies specific for IL2 (MQ1-17H12), IFNγ (4S.B3) and TNFα (Mab11) were all purchased from Pharmingen (Hamburg, Germany), as were irrelevant isotype specific antibodies, unlabeled anti-cytokine antibodies and recombinant cytokines. Paraformaldehyde (PFA) and saponin were obtained from Riedel de Haen (Seelze, Germany). PMA, ionomycin and monensin were purchased

Cytokine expression after 5 h of stimulation with PMA and ionomycin

Cytokine expression in CD3+, CD3+CD8−, CD3+CD8+, CD45RA+ and CD45R0+ cells was investigated in 50 healthy blood donors after 5 h stimulation with PMA and ionomycin. Cytometric profiles of one representative individual are shown in Fig. 2, while means±SD of all 50 individuals are given in the text.

32.8±10.4% of all T-cells produced IL2 (Fig. 2a). Subdividing T-cells into CD8+ and CD8- cells, CD8- cells proved to be the main contributors to IL2 production (27.9±9.9%) while only a few CD8+ cells

Discussion

Simultaneous staining of cytokines and surface markers showed that lymphocyte subpopulations differ quite markedly in their cytokine production. While IL2 and TNFα were mainly produced by T helper cells (CD3+CD8−), CD3+CD8+ cells mostly produced IFNγ and only low percentages were positive for IL2 and TNFα. This has already been shown using T cell clones of the cytotoxic or helper type (Salgame et al., 1991), but intracellular staining using PBMC has the advantage that it estimates relative

Acknowledgements

We would like to thank Anja-Maria Thurau and Veronika Baumeister in the group of Prof. C.H.L. Rieger and PD U. Schauer (Bochum, Germany) for guiding us in the initial stages of cell preparation for the flow cytometric analysis of intracellular cytokines. We also would like to thank Sabine Nissen for her help in preparing PBMC for flow cytometric analysis.

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