Abstract

Cytokines are a group of proteins that mediate complex immune cell interactions and regulate the development of effector immune responses. The accurate measurement of cytokines has broad-ranging applications for characterizing inflammation¹, and studying pathogen-specific immune responses.^2,3 Unfortunately, conventional cytokine assays are invariably species-specific, and therefore difficult to adapt for use in marine mammals. Furthermore, the production of reagents for measuring secreted cytokines is expensive, time-consuming and labor intensive. Real-time TaqMan® PCR (Applied Biosystems, Foster City, CA) is a new and promising method for quantifying RNA and DNA by measuring PCR product accumulation through a dual-labeled fluorogenic TaqMan® probe. Recent applications of this technology to the measurement of cytokine mRNA levels have provided reliable and affordable estimates of cytokine production⁴. In this study the nucleotide sequence alignments of cytokines from several species, including partial-length cetacean sequences⁵, were used to design degenerate primers. In addition, previously published canine and bovine cytokine primers were constructed and used to amplify cytokine sequences from 3 California sea lions (Zalophus californianus) and 2 bottlenose dolphins (Tursiops truncatus), respectively. Cytokine mRNA production was stimulated in vitro by exposing peripheral blood mononuclear cells (PBMCs) to either 5 μg/ml of pokeweed mitogen (PWM), or concanavalin A (con A) for 12 to 24 hours. Amplification of individual cytokine sequences was achieved by PCR performed on random hexamer-generated cDNA from these stimulated PBMCs. The amplified sequences were used to design species-specific primers and an internal oligonucleotide probe for each cytokine of interest. The probe was labeled with a 5' reporter and a 3' quencher dye and the conditions for real-time PCR were optimized for each cytokine gene. An internal control (calibrator) gene (either GAPDH or 18S rDNA) was used to normalize for RNA quantity and quality, and for the efficiency of the RT and PCR reactions. The quantities of cytokine RNA in all the samples were therefore expressed as an n-fold difference relative to the calibrator (internal control).

Partial length species-specific sequences were obtained for the cytokine genes encoding IL-2, IL-4, IL-6, IL-10, IL12p40, and IFN-γ in the dolphin, and IL-2, IL-10, and IL12p40 genes in the California sea lion. In both species real time quantitative PCR was successfully performed for each of these cytokines (Figures 1 and 2) except IL-2. The cytokine signals were successfully normalized using internal control genes from each species. The specificity of the assays was confirmed by sequencing the amplified products.

These results show that the application of real-time RT-PCR offers an affordable, sensitive, and readily applicable method for quantifying marine mammal cytokine gene expression in peripheral blood samples. Several studies have shown that alterations in cytokine levels are associated with a variety of immune-mediated and inflammatory disorders^2,6. The successful adaptation of these sensitive and highly reproducible assays to two major marine mammal species represents a major advance in our ability to accurately quantify pathogen-specific immunological responses, and to detect specific and polarized changes in inflammatory responses in these species. Future studies will concentrate on expanding the range of specific cytokine reagents for this assay, particularly in the California sea lion, and to apply this methodology in controlled clinical studies.

Figures

 Acknowledgements

The authors wish to thank the Office of Naval Research for providing financial support for this project.

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