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Clinical and Diagnostic Laboratory Immunology, November 2000, p. 920-924, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Measurement of Cytokine Secretion, Intracellular Protein Expression, and mRNA in Resting and Stimulated Peripheral Blood Mononuclear Cells

Kathleen E. Sullivan,* Joie Cutilli, Lisa M. Piliero, Darlene Ghavimi-Alagha, Stuart E. Starr, Donald E. Campbell, and Steven D. Douglas

Division of Immunologic and Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Received 25 February 2000/Returned for modification 25 May 2000/Accepted 13 August 2000

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.

* Corresponding author. Mailing address: Immunologic and Infectious Diseases, The Children's Hospital of Philadelphia, 34th St. and Civic Ctr. Blvd., Philadelphia, PA 19104. Phone: (215) 590-1697. Fax: (215) 590-3044. E-mail: sullivak{at}mail.med.upenn.edu.

Clinical and Diagnostic Laboratory Immunology, November 2000, p. 920-924, Vol. 7, No. 6
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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