Flow cytometric analysis of intracellular IFN-γ, IL-4 and IL-10 in CD3+4+ T-cells from rat spleen

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Abstract

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3+4+ T-cells that produce IFN-γ, IL-4 and IL-10. In vitro stimulation of IFN-γ production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.

Introduction

T-cells and other immune cells exert their effector function partly through the production and release of cytokines. Th1 and Th2 cells are characterised by their distinct cytokine patterns (Mosmann and Coffman, 1989). Th1 cells secrete IFN-γ and IL-2 whereas Th2 cells produce IL-4, IL-5 and IL-10. Various methods are currently employed to detect cytokine production and secretion. Measurement of cytokines can be done using ELISA systems which are highly specific but measure only secreted cytokine, i.e. reflecting the net outcome of produced, absorbed and degraded cytokine. This type of analysis does not measure cytokine production at the single cell level. Various methods for assessing cytokine expression and production at the single cell level have been reviewed (Lewis, 1991). Cytokine expression in T-cells has been analysed by RT-PCR but the limitation with this type of analysis is that it does not give information about production of different cytokines by individual cells nor does it allow one to study the contribution of different cells to cytokine production in a heterogeneous cell population. Based on an adaptation of the fixation and permeabilisation method described by Sander et al. (1991), several groups have reported the use of multi-parameter flow cytometry for assessment of single-cell intracellular cytokine production in human PBMCs (Jung et al., 1993, Elson et al., 1995, Mascher et al., 1999) and in mouse T-cells (Assenmacher et al., 1994, Kerttula et al., 1999). In a recent article Prussin (1997) outlined the main strengths of intracellular flow cytometry, which include the ability to distinguish between Th1 and Th2 cells, examine cytokine production in rare cell populations and also the ability to analyse cytokine production at the level of individual cells. The availability of directly conjugated, fluorescent anti-cytokine antibodies has greatly enhanced the sensitivity of detection to levels not previously available when using indirect staining procedures. However, such antibodies are widely available for human and mouse, but not for rat. Previous flow cytometry studies investigating intracellular cytokines in rat tissues have been reported (Berguer and Ferrick, 1995, Bernard et al., 1998, Klemm et al., 1998) and involved analysing expression of IFN-γ in T-cells by dual colour flow cytometry. Here we report on the use of three-colour flow cytometry for detection of rat T-cells that produce IFN-γ, IL-4 and IL-10 cytokines in cultured splenic T-cells that were obtained from a substrain of the Wistar rat, the BB control (non-diabetes-prone) rat.

Section snippets

Monoclonal antibodies and reagents

Mouse anti-rat monoclonal antibodies: CD3 FITC and NA/LE (G4.18), CD4 Cy-Chrome® (OX-35), CD28 NA/LE (JJ319), IL-4 PE and purified (OX-81), IL-10 PE and purified (A5-4), recombinant rat IL-4, rat anti-mouse CD4 FITC (RM4-5), rat anti-mouse IFN-γ PE (XMG1.2), recombinant mouse IL-2, immunoglobulin isotype controls: mouse IgG1κ PE (MOPC-21), mouse IgG2b PE (27-35). MiCK-1 positive control cells, RiCK-2 positive control cells, Cytofix/Cytoperm™ Kit, GolgiStop®, GolgiPlug™, were obtained from BD

Optimisation of staining protocol

Of the antibody concentrations, incubation temperatures and duration of incubation examined, the following optimal conditions were established. For cell surface staining, the cells were incubated with 0.5 μg CD3 FITC antibody and 0.5 μg CD4 Cy-Chrome® antibody for 10 min at room temperature (data not shown). The optimal concentrations for each anti-cytokine antibody were found to be 0.25 μg IFN-γ PE, 0.25 μg IL-4 PE and 0.1 μg IL-10 PE. Isotype controls (IgG1κ PE for both IFN-γ and IL-4; IgG2b

Discussion

We have used intracellular staining for cytokines, combined with flow cytometry, to examine the frequencies of cytokine producing cells from rat spleen tissue. Over the past 10 years, the evolution of multiparameter flow cytometry has permitted the detection of one or more cytokines within an individual cell, permitting analysis of simultaneous production of various cytokines by a heterogeneous cell population.

The primary objective of our study was to modify the intracellular flow cytometric

Acknowledgements

We thank Dr. David Ernst of BD PharMingen for reviewing the manuscript. We would like to thank Jocelyn Souligny and Dominique Patry for supplying and caring for the animals, Judy Edgar for technical assistance, and Pierre Thibert, Chief of the Animal Resources Division, for maintaining the rat colony. These studies were supported by the Medical Research Council of Canada, The Ottawa Hospital Research Institute, The Ontario Research and Development Challenge Fund, Health Canada and BD PharMingen.

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