Quantification of human interleukin 18 mRNA expression by competitive reverse transcriptase polymerase chain reaction

Area of Science:

• Immunology
• Molecular Biology
• Cytokine Research

Background:

• Interleukin 18 (IL-18), initially known as interferon gamma inducing factor, is a cytokine.
• IL-18 induces interferon gamma production in Th1 cells.
• IL-18 is expressed in various cell types, including mononuclear phagocytes, osteoblasts, keratinocytes, and adrenal cortex cells.

Purpose of the Study:

• To develop a method for quantifying human IL-18 mRNA expression in small cell samples.
• To assess basal and stimulated IL-18 mRNA expression in different peripheral blood mononuclear cell (PBMC) subtypes.

Main Methods:

• Development of a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay using a competitive template.
• Quantification of human IL-18 mRNA expression in primary peripheral blood monocytes, CD4(+)T cells, CD8(+)T cells, B cells, and NK cells.
• In vitro stimulation of PBMC from healthy donors with monocyte-specific (lipopolysaccharide, LPS), T-cell-specific (anti-CD3), and polyclonal (phytohaemagglutinin, PHA) stimuli.

Main Results:

• The developed competitive RT-PCR assay is a valid, sensitive, and precise tool for quantifying IL-18 mRNA.
• Basal IL-18 mRNA expression was detected in all tested PBMC subtypes.
• Only LPS stimulation led to a significant increase in IL-18 mRNA expression, peaking at 2 hours, while anti-CD3 and PHA showed minimal effects.

Conclusions:

• IL-18 is constitutively expressed by all major PBMC subtypes.
• Activated monocytes, particularly during inflammation, are likely the main source of IL-18 expression due to significant induction by LPS.
• The study provides a reliable method for quantifying IL-18 mRNA and insights into its expression regulation in immune cells.