Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology

https://doi.org/10.1016/S0022-1759(98)00079-9Get rights and content

Abstract

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFα, IL-2, IL-4, IL-6, IL-10, and IFNγ. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNγ mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFα or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Introduction

Cytokines are low molecular weight proteins that regulate both cell-mediated and humoral immune responses. Given their central role in immunomodulation, cytokines may be useful as indicators of the presence and severity of disease. Different patterns in the expression of cytokines have been associated with a wide variety of clinical disorders such as AIDS (Ameglio et al., 1994; Fan et al., 1993), autoimmune disorders (De Carli et al., 1994), rheumatoid arthritis (Elliott et al., 1995; Katsikis et al., 1994), and allograft rejection (Gaston, 1994).

Numerous assays have been used to assess cytokine levels in clinical specimens. Bioassays measure only biologically active cytokines (Mire-Sluis et al., 1995b), whereas immunoassays detect both biologically active and inactive cytokines. However, a number of difficulties associated with use of immunoassays to measure cytokine levels in clinical specimens have been reported, including the presence of various cytokine binding proteins, such as soluble cytokine receptors, that can affect the recognition of cytokines by immunoassay, as well as the methods used for sample preparation, storage, and analysis that can influence the validity of cytokine measurements (Mire-Sluis et al., 1995a). As an alternative to assays for detection of cytokine protein, nucleic acid-based assays for the detection and quantification of cytokine mRNA have been used successfully to assess cytokine expression in patient specimens (for example, Hockett et al., 1995). Assays based on the polymerase chain reaction (PCR) offer exquisite sensitivity for detection of cytokine mRNA, but these assays are plagued by the vagaries of specimen processing and require numerous precautions to avoid contamination of specimens with PCR products and carryover from other patient specimens (Persing, 1991). Clearly, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting.

The branched DNA (bDNA) assay represents a significant advance in the quantification of nucleic acid molecules for research and clinical applications. Fundamentally different from target amplification methods such as PCR, the bDNA assay measures nucleic acid molecules by boosting the reporter signal, rather than amplifying target sequences as the means of detection, and hence is not subject to the errors inherent in the extraction and amplification steps of PCR-based methods. The bDNA assay has been applied to the direct quantification of several viral nucleic acids, including hepatitis C virus RNA (Detmer et al., 1996), hepatitis B virus DNA (Hendricks et al., 1995), human immunodeficiency virus type-1 RNA (Pachl et al., 1995), and human cytomegalovirus DNA (Chernoff et al., 1997). Inherently quantitative and requiring minimal sample preparation, the bDNA assay also may prove useful for measuring low abundance transcripts in clinical specimens. For example, the bDNA assay recently has been applied to quantification of insulin mRNA in pancreas islets (Wang et al., 1997).

Here, we report the development of a bDNA assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). We describe the design of the assay and verify the specificity of the oligonucleotide probes. We assess the sensitivity, linearity, and reproducibility of the bDNA assay, and explore specimen storage conditions that may affect cytokine mRNA quantification. Using relative light units (RLUs) as the unit of measurement, we evaluate relative amounts of cytokine mRNAs and demonstrate the potential clinical utility of the bDNA assay by monitoring cytokine mRNA levels in healthy blood donors and comparing cytokine mRNA levels in PBMCs from HIV-1 seropositive vs. HIV-1 seronegative subjects.

Section snippets

Clinical specimens

Blood specimens were obtained from HIV seronegative healthy laboratory personnel (at Chiron Corporation) and from HIV-1 seropositive subjects participating in the UCLA Multi-center AIDS Cohort Study (MACS). All MACS participants are homosexual men for whom detailed histories are available (Kaslow et al., 1987).

Preparation of PBMCs

Blood was collected into EDTA anticoagulant tubes and processed within the next 2 hours. PBMCs were isolated using either Leucoprep tubes containing sodium citrate (No. 2761, Becton

Principles of the bDNA assay for cytokine mRNA quantification

A schematic diagram of the bDNA assay for cytokine mRNA quantification is shown in Fig. 1. Similar to an ELISA in its basic approach, the bDNA assay uses a solution phase sandwich assay format in which cytokine mRNA is hybridized in solution with oligonucleotide target probes. The cytokine mRNA with bound target probes is captured onto oligonucleotide-modified microwells, and then is hybridized with bDNA amplifier molecules. Amplification of the cytokine mRNA is achieved by the binding of

Discussion

Quantification of cytokine expression in clinical specimens, both at the level of protein and mRNA, has been used to understand how cytokine response is correlated with the rate of disease progression and the degree of immunologic deterioration. In this study, we have evaluated the performance of the bDNA assay for quantification of several human cytokine mRNAs, including TNF-α, IL-2, IL-4, IL-6, IL-10, and IFN-γ. Unlike target amplification methods such as RT-PCR, the bDNA assay uses signal

Acknowledgements

We thank David Grounds for support in tissue culture, Dubravka Opuhac and Kristina Whitfield for graphics, and Dr. Linda Wuestehube for writing and editorial assistance. This work was supported in part by NIH grants TW 00003, AI 36086 and AI 35040.

References (38)

  • D.N. Chernoff et al.

    Quantification of cytomegalovirus DNA in peripheral blood leukocytes by a branched-DNA signal amplification assay

    J. Clin. Microbiol.

    (1997)
  • M.L. Collins et al.

    A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml

    Nucleic Acids Res.

    (1997)
  • M. De Carli et al.

    Human Th1 and Th2 cells: functional properties, regulation of development and role in autoimmunity

    Autoimmunity

    (1994)
  • W. Degrave et al.

    Cloning and structure of the human interleukin 2 chromosomal gene

    EMBO J.

    (1983)
  • J. Detmer et al.

    Accurate quantification of HCV RNA from all HCV genotypes using branched DNA (bDNA) technology

    J. Clin. Microbiol.

    (1996)
  • C.A. Dinarello et al.

    Cytokine measurements in septic shock

    Ann. Intern. Med.

    (1993)
  • J. Fan et al.

    Elevated IFN alpha and decreased IL-2 gene expression are associated with HIV infection

    J. Immunol.

    (1993)
  • T. Fujita et al.

    Structure of the human interleukin 2 gene

    Proc. Natl. Acad. Sci. U.S.A.

    (1983)
  • R.S. Gaston

    Cytokines and transplantation: a clinical perspective

    Transplant Sci.

    (1994)
  • Cited by (10)

    • Genomics and proteomics: The new millennium of drug discovery and development

      2000, Journal of Pharmacological and Toxicological Methods
    • Signal amplification-based techniques

      2016, Nucleic Acid Testing for Human Disease
    View all citing articles on Scopus
    View full text