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A multisystem approach to cytokine research
Daniel P. Collins
Multiplexed flow cytometry offers an innovative tool for monitoring and quantitating cytokine levels in biological samples.
Researchers and clinicians are increasingly looking for soluble indicators that can provide clues to immune function or be used as markers of disease progression in immune disorders, transplantation medicine, and infectious diseases. Among the markers that have attracted the attention of researchers are the cytokines, small peptide proteins with hormone-like activity that are secreted by cells of the immune system in response to cell activation and the initiation of the immune response.
Cytokines play a central role in communication among cells of the immune system. They are soluble mediators and regulators of innate and specific immunity.1–4 Additionally, cytokines promote the growth and differentiation of leukocytes and blood-cell precursors. They are central agents in the activation and proliferation of antigen-specific immune cells, activities that are among the earliest steps on the way toward developing an immune response.4
This article discusses the relationship of cytokines to immune function, the potential of cytokines as soluble indicators of immune function and disease progression, and the need for a comprehensive approach to study them. Additionally, the article examines the advantages of flow cytometry as a tool for a multisystem study of cytokine biology.
Cytokine Biology
Because of the interplay among cytokines and the cells that respond to their biological activity, understanding cytokine biology requires study of changes in the plasma levels of soluble cytokines, changes in cell-surface cytokine receptor expression, and the expression of intracellular cytokines by individual cell subpopulations.
Changes in plasma levels of cytokines, or in the ability of cells to produce or respond to cytokines, can be used as in vitro indicators of immune dysfunction or disease progression.5–7 High levels of such inflammatory cytokines as Interleukin-1 (IL-1), Interleukin-6 (IL-6), and tumor necrosis factor (TNF-
) can be indicators of sepsis. Various clinical conditions (including HIV) have been shown to reduce both cytokine production and cytokine-receptor expression.7 Overexpression of receptors for Interleukin-2 (IL-2) on circulating T-cells can be an indicator of graft rejection.
Even when no cytokines can be detected in the plasma, changes in the expression of cell-surface cytokine receptors can provide an indirect indication of cytokine activity. Cytokines interact with cells by means of specific cell-surface receptors. When leukocytes become activated, the number of their cell-surface receptors for stimulatory cytokines increases.1 This causes the cells to become more responsive to those cytokines. As part of a positive-regulation loop, cytokines can also up regulate their own receptors on target cells. Circulating leukocytes often show the effects of cytokines and of activation by up-regulation of cell-surface cytokine receptors. Observing changes in cellular cytokine receptor expression could provide clues to immune involvement in particular pathologic conditions.
Activated leukocytes are not only more receptive to cytokines, they also produce cytokines. These cytokines have autocrine activity—increasing the proliferation, differentiation, or effector function of their own cell subset. They may also have far-ranging effects on other cell types. For example, the main orchestrators of immune response, T-helper lymphocytes, are subdivided into Thelper1 and Thelper2 subsets according to the range of cytokines they secrete. Thelper1 cells mainly secrete the cytokines that promote cellular immunity—IL-2 and Interferon-gamma (IFN-
). Thelper2 cells secrete the Interleukins IL-4, IL-5, and IL-10, which direct the body toward a more humoral (antibody-mediated) immune response. Development of Thelper1-dependent immunity has been considered to be protective against the infectious agents of several diseases (e.g., leishmaniasis and HIV). Development of Thelper2-dependent humoral immunity protects the host against many bacterial and viral infections, but may actually protect the infectious agent from a successful host immune response in certain parasitic and viral infections. Using cytokine secretion as a means of distinguishing and monitoring Thelper1 and Thelper2 immune responses could prove important for the development of effective vaccines.
Assay Technologies
In the past, mitogenic assays have been used to measure lymphocyte activation as an indicator of immune function. Mitogenic assays measure the proliferative response of isolated mononuclear cells to in vitro stimulation by mitogenic lectins (phytohemagglutinin, concanavalin A, pokeweed mitogen) or by certain specific antigens (streptokinase, PPD).
The index of activation is a proportion determined by the relative uptake of radiolabeled nucleotides (3H-thymidine) by the mitogen-stimulated culture compared to a basal nonstimulated culture. Actively proliferating cells incorporate more radionucleotides than weakly proliferating cells. Nonproliferating cells should incorporate few radionucleotides, if any.
Performing a mitogenic assay can take between 48 to 72 hours, and requires licensing, storage, and disposal of radioactive waste. A similar flow-cytometry-based assay measures the uptake of the nonradioactive nucleotide bromodeoxyuridine (BrdU), as detected with a fluorescent anti-BrdU antibody. These assays are somewhat nonspecific and provide little information regarding cytokine production or cell communication.
These mitogenic tests have recently been supplanted by assays that use flow cytometry to measure changes in cell-surface markers and the expression of intracellular cytokines. Flow cytometers are laser-based cell counters that can distinguish different cell populations by their differing light-scattering characteristics. Depending on the model, a flow cytometer may also be able to distinguish the emissions of three or more different fluorescent antibodies. Such antibodies are created by attaching dyes with distinct fluorescent emissions to monoclonal antibodies that recognize specific cell-surface antigens. When mixed with a biological sample, these fluorescent antibodies bind to their target cells, if present, which enables them to be counted by the flow cytometer.
By combining the flow cytometer's abilities to distinguish particles either by their light scatter or their fluorescence, researchers can develop multiplexed assays that provide more information with fewer tests. Combining the differences in light-scatter characteristics with differences in the binding of fluorescently labeled antibodies is the basis for immunophenotyping.
Cellular Analysis
The presence or absence of cytokine receptors on cell surfaces can provide information about their state of activation. Cytokine receptors can be analyzed by flow cytometry using either fluorescently labeled antireceptor antibodies or fluorescently labeled cytokines. Combining these reagents with antibodies against CD markers enables analysis of cell activation within specific cell populations (see Figure 1).
Figure 1. Expression of functional cell-surface receptors for Interleukin-2 on CD4 T-cells after 60 hours without activation (a) and with activation by anti-CD3 and anti-CD28 antibodies (b). The cell populations positive for both CD4 and the IL-2 receptor are highlighted in green. Cells are identified using FITC-labeled anti-CD4 antibody and the phycoerythrin-labeled human cytokine Interleukin-2.
T-lymphocyte activation is associated with an up-regulation of cell-surface receptors for the cytokine IL-2. IL-2 promotes the multiplication and development of effector function in T- and B-lymphocytes and is critical to the immune response. Since circulating T-cells do not normally express IL-2 receptors, such expression can be indicative of immune activation or an ongoing pathology. The inability of certain T-cell subsets to express IL-2 receptors after activation may indicate defects in immunity.
Cells that secrete cytokines can also be studied by flow cytometry.8 Lymphocytes are labeled with anti-CD antibodies to identify cells by their subset. The cells are stabilized by fixation with formaldehyde, and a detergent is then used to create holes in the cell membranes so that anticytokine antibodies can pass into the interiors of the cells. Cells that are producing and secreting cytokines express intracellular cytokines that will bind the fluorescently labeled anticytokine antibodies and be counted by the flow cytometer.
Activated T-lymphocytes can be subdivided into several different populations according to their staining characteristics, and analyzed by means of three-color flow cytometry. For example, populations of CD4+ and CD4– cells can be identified using an FITC-labeled anti-CD4 antibody, which labels the cell surface. IL-4 producing and IL-4 nonproducing cell populations can be identified by intracellular labeling with a Cy5PE-labeled anti-IL-4 antibody. And IFN- producing and nonproducing cell populations can be identified by intracellular labeling with PE-labeled antiInterferon antibody (see Figure 2). This procedure can help researchers differentiate between Thelper1 (IFN- producing) and Thelper2 (IL-4 producing) cells. Similarly, any PE- or Cy5PE-labeled anticytokine antibody could be paired with an appropriate FITC-labeled anti-CD antibody to identify intracellular cytokines in specific cell populations.
Figure 2. Isolated T-lymphocytes were cultured for 60 hours in either the presence or absence of activation with anti-CD3 and anti-CD28 antibodies. Unactivated (a) and activated (c) cells labeled for CD4 and intracellular IFN-. Unactivated (b) and activated (d) cells labeled for CD4 and intracellular IL-4. The cell surface was labeled with anti-CD4-FITC antibodies. Intracellular cytokines were detected with anti-IL-4 Cy5PE and anti-IFN- PE-labeled antibodies. Cells that express both CD4 and either IFN- or IL-4 are highlighted in green in the above histograms.
Quantitation of Cytokine Secretion
Cytokine levels in biological samples can be quantitated indirectly by using a bioassay to measure their biological activity. Researchers can also quantitate cytokine levels directly using enzyme immunoassays, radioimmunoassays, or chemiluminescence assays. Although the effective analytical range of such assays is large (15–2000 pg/ml), the lower limit of detection is still well above normal serum levels for cytokines. In addition, such assays can only quantitate one cytokine at a time.
To improve on the performance of current methods of quantitation, BioE (St. Paul, MN) has developed immunoassays that make use of the multiplexing abilities of flow cytometry to simultaneously quantitate three cytokines in a single sample. These assays have a wider dynamic range than current methods (0.5–20,000 pg/ml), making them capable of detecting normal serum levels of many cytokines.
The system uses 7-µm-diam paramagnetic particles labeled with a monoclonal antibody specific to a particular cytokine. These particles capture the target cytokines from the fluid phase of a biological sample. The presence of the captured cytokine is reported by means of a fluorescently labeled antibody that binds to a region of the cytokine distinct from the site targeted by the capture antibody. If the cytokine of interest is present in the biological sample, the capture particle will fluoresce with an intensity directly proportional to the concentration of the cytokine in the sample.
A multiplexed cytokine assay can be developed by combining several types of particles, each targeted to a different cytokine, with fluorescent reporter antibodies specific to those same cytokines. Each reporter antibody is labeled with a different fluorescent dye, making it possible to simultaneously detect and distinguish multiple cytokines (see Figure 3). To quantitate cytokine levels, the researcher compares the intensity of the assay's fluorescent staining to that of a standard curve created with known concentrations of the cytokine (see Figure 4).
Figure 3. Schematics of the Multiflow multiplexed cytokine immunoassay. Separate particles are
coated with antibodies specific for a single cytokine. These particles then capture the soluble cytokines from biological fluids. Fluorescently labeled reporter antibodies allow detection of the captured cytokine. In this instance, IFN- is reported with a fluorescein-labeled antibody resulting in green fluorescence. IL-2 is reported with a phycoerythrin-labeled antibody resulting in orange fluorescence. IL-4 is reported with a Cy5PE-labeled antibody resulting in a red fluorescence. Intensity of the fluorescent signal in each individual color range is directly proportional to the concentration of the cytokine in the sample.
Technologies for studying cytokinesBioassays. Bioassays are in vitro culture systems using cytokine-dependent cell lines. They determine the specific biological activity of a cytokine-containing medium by stimulating growth of the dependent cell line. Proliferation induced by the unknown sample is compared to standards of a known concentration of cytokine. The proliferation index is determined by measuring the cellular uptake of 3H-thymidine. These assays use radionucleotides that require licensure, storage, and disposal. Although sensitive, they require 24–72 hours of in vitro culture time, and measure only the biologically available forms of the cytokine. Enzyme Immunoassays. Usually provided in a 96-well plate format, these assays are based on the capture of cytokines by monoclonal antibodies attached to the surface of each well. A biotin-labeled second antibody directed against the cytokine is reacted with the wells to detect the presence of the cytokine. Streptavidin-peroxidase is reacted with the well and binds to the biotin-labeled antibody. The peroxidase enzyme catalyzes a colorimetric reaction that can be measured as a function of optical density at 450 nm. These assays measure one cytokine at a time, in the range of 15–2000 pg/ml, and require four to five hours to perform. Although such assays are sensitive enough to measure tissue culture levels of most cytokines, most enzyme immunoassays are not sensitive enough to detect normal serum levels. Flow Cytometry Bead IFA. This new technology uses the multiplexing capacity and high sensitivity of flow cytometry to quantitate soluble analytes. Captured analyte particles are reported by fluorescently labeled antibodies. This technology shows a high potential for both improved sensitivity and multiplexability. Measurement of Cytokine Receptors by Flow Cytometry. This assay makes use of competitive binding of antireceptor antibodies or direct binding of fluorescently labeled cytokines to cells. It can measure relative up- and down-regulation of cytokine receptors in mixed cell populations by using anti-CD antibodies. Measurement of Cytokine Receptors by RIA. Competitive binding of 125I-cytokine with unlabeled cytokine. This assay can find an average number of cytokine receptors per cell over the entire population of cells and determine the affinity coefficient of binding for the cytokine. However, it cannot determine differences in binding between different populations in a mixed cell culture. Measurement of Intracellular Cytokines. A flow cytometry assay. Cytokine-producing cells are stabilized by formaldehyde fixation and made permeable by detergent. Fluorescent-labeled anticytokine antibodies are reacted with the permeable cells. Cells producing the cytokine will become fluorescent. These assays require a cell activation step and in vitro culture. Although they can determine which populations of cells are producing the cytokines and qualitatively assess relative levels of cytokine production, they cannot quantitate cytokine production per cell. Measurement of mRNA. Posttranscriptional mRNA can be measured by PCR, Northern blot analysis, or colorimetric assays. This method offers strong correlation to protein production, but no information about which cells are responsible for that production. |
When analyzed by flow cytometry, the particles used in the Immunoflow-IFA and Multiflow-IFA assays have light-scattering properties that differ from those of human leukocytes. This enables the particles to be analyzed in the same sample simultaneously with cells, providing a unique tool to study cells and the proteins they secrete.
Figure 4. Development of standard curve with the Multiflow-IFA assay. Beads are incubated with
prediluted cytokine standards, washed, and reacted with three-color reporter antibody. The beads are gated by light scatter (a). Fluorescence intensity for each positive peak is recorded for each standard; green fluorescence correlates to IFN- concentration (b); orange fluorescence correlates to IL-2 concentration (c); red fluorescence correlates to IL-4 concentration (d). The fluorescence peaks for each standard of IL-2 are overlaid (e). When plotted as mean fluorescence intensity versus concentration of cytokine, the standard curves for IFN-, IL-2, and IL-4 are developed (f, g, h).
Combining results from the cellular analysis and quantitative secretion assays provides powerful information. Lack of response to an infectious agent could result from a reduction in overall cytokine production, a reduction in the number of cells producing cytokines, a lack of expression of cytokine receptors, or a defect in a specific subpopulation of responding cells. By combining cellular analysis and quantitation of secretion, researchers can determine the average number of molecules secreted per cytokine-producing cell, and thus gain a better understanding of the reasons for the unresponsiveness of a patient's immune system.
Ongoing studies are investigating the differences in T-cell activation in individuals with active viral infections. T-cell enriched lymphocytes were obtained from peripheral blood samples of normal individuals and individuals with various viral infections. The T-cells were activated in vitro with anti-CD3 antibody, IL-1, and IL-2. After 20 hours the cells and culture supernatants were collected. The supernatants were analyzed for secretion of IFN-, IL-2, IL-4, and GM-CSF and the cells were analyzed for the presence of IL-2 receptors and intracellular IFN-, IL-2, IL-4 and GM-CSF. No significant differences were found in the cellular component of the analysis. However, it was found that activated T-cells from individuals with active viral infections consistently secreted less GM-CSF than cells from normal individuals. Results are presented in Table I.
| Sample | 20-hour Accumulation (pg/ml) | Secretion per Hour pg/ml/106 cells |
|---|---|---|
| Normal | 80–250 | 4.0–8.0 |
| HTLV 1 | 35–38.5 | 0.8–2.3 |
| HBV surface | 4.1–15 | 0.28–0.5 |
| HBV core | 80–100 | 4.7–5.6 |
| Influenza (active) | 42.5 | 3.8 |
| Influenza (postrecovery) | 71 | 4.0 |
| CMV | 27–30 | 1.1–1.8 |
| 2-way MLR | 48.5 | 3.4 |
Table I. Effects of viral infection on secretion of GM-CSF by CD3-activated T-cells.6
Conclusion
Cytokines provide a useful tool to study T-cell activation and immune function and may provide a focus for the development of new immune-based diagnostics. T-cell activation is a critical first step in the development of immune function, initiating a complex cascade of events that includes up-regulation of cytokine receptors, cellular proliferation, and the production and secretion of cytokines. In turn, these initial cytokines can stimulate the production and secretion of other cytokines, leading to the recruitment and activation of effector cells.
Studying interactions of cytokines with cells of the immune system can help delineate defects in immune function. Our understanding of the role of cytokines in lymphocyte activation can be improved by technologies that permit simultaneous monitoring of cell-surface molecules, cytokine receptors, and the expression of internal cytokines, while also quantitating cytokine secretion.
The multiplexing capacity of flow cytometry is being explored as a means for simultaneous analysis of cells and the cytokines they produce. The added sensitivity and analytical range offered may enable researchers to determine the normal ranges of certain cytokines in plasma. Such assays can have a direct clinical impact when applied to the study of T-cell function in HIV and other viral infections, autoimmunity, and transplantation immunology.
References
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2. F Herrmann et al., "Interleukin 1 Stimulates T Lymphocytes to Produce Granulocyte-Monocyte Colony Stimulation Factor," Journal of Clinical Investigation 81 (1988): 1415–1418.
3. D Lovett et al., "Interleukin 1 as a Mediator of Tumor Cytostasis," Journal of Immunology 136 (1986): 340–347.
4. KA Smith, "T-Cell Growth Factor," Immunology Review 51 (1980): 337–357.
5. DP Collins, BJ Luebering, and DM Shaut, "T-Lymphocyte Functionality Assessed by Analysis of Cytokine Receptor Expression, Intracellular Cytokine Expression, and Femtomolar Detection of Cytokine Secretion by Quantitative Flow Cytometry," Cytometry 33 (1998): 249–255.
6. DP Collins, "Multi-System Approach to Analysis of T-Lymphocyte Activation by Flow Cytometry: Utilization of Intracellular Cytokine Expression, Cytokine Receptor Expression, and Quantification of Cytokine Secretion as an Indicator of Activation," Clinical Immunology Newsletter 18, no. 11/12 (1998): 140–145.
7. J Yoo et al., "Altered Cytokine Production and Accessory Cell Function after HIV Infection," Journal of Immunology 157 (1996): 1313–1320.
8. C Prussin and DD Metcalfe, "Detection of Intracytoplasmic Cytokine Using Flow Cytometry and Directly Conjugated Antibodies," Journal of Immunological Methods 188 (1995): 117–
128.
Daniel P. Collins, PhD, MSPH, is executive vice president and chief scientific officer at BioErgonomics Inc. (St. Paul, MN).
