Measurement of cytokine secretion, intracellular protein expression, and mRNA in resting and stimulated peripheral blood mononuclear cells

Clin Diagn Lab Immunol. 2000 Nov;7(6):920-4. doi: 10.1128/cdli.7.6.920-924.2000.

Abstract

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-alpha), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-alpha, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Cytokines / biosynthesis*
  • Cytokines / genetics*
  • Cytokines / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Gene Expression
  • Humans
  • In Vitro Techniques
  • Interferon-gamma / biosynthesis
  • Interferon-gamma / genetics
  • Interferon-gamma / metabolism
  • Interleukin-2 / biosynthesis
  • Interleukin-2 / genetics
  • Interleukin-2 / metabolism
  • Interleukin-4 / biosynthesis
  • Interleukin-4 / genetics
  • Interleukin-4 / metabolism
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / immunology*
  • Leukocytes, Mononuclear / metabolism*
  • Lymphocyte Activation / drug effects
  • Middle Aged
  • Phytohemagglutinins / pharmacology
  • Polymerase Chain Reaction
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Tumor Necrosis Factor-alpha / biosynthesis
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Cytokines
  • Interleukin-2
  • Phytohemagglutinins
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • Interleukin-4
  • Interferon-gamma
  • Tetradecanoylphorbol Acetate