by Catherine Shaffer
Cytokines are a large family of molecules that regulate the immune system. They are created in response to a stimulus such as inflammation and deliver messages that regulate growth and differentiation of immune cells. Cytokines can be therapeutic targets for drug development, or they can be therapies themselves. Cytokines are also of great interest in basic research, helping scientists untangle complex signaling pathways. Because of this role, they are also prime candidates for biomarker discovery.
One of the most common techniques for analyzing cytokines is the microarray. Customized cytokine sets are available through many vendors of commercial microarrays. These arrays are powerful for picking up protein interactions involving cytokines or increased expression of cytokines, and are capable of handling a large number of cytokines at one time. Increased multiplexing is an emerging need in the field, where it is increasingly clear that understanding the behavior of a single cytokine in isolation is not very useful, but capturing a snapshot of the behavior of a dozen or even a hundred cytokines at once could be extremely revealing.
Real-time PCR has emerged as a newer alternative to the standard antibody microarray, and can be used for powerful effect in certain situations. Because some cytokines are low abundance molecules, they can be difficult to detect. Real-time PCR technology has a wide linear dynamic range that is sensitive at very low levels of abundance.
One such technology is the RT2 Profiler Array available from SABiosciences. The array contains 96 or 384 optimized RT-PCR assays including controls for related pathways or diseases, including a number of cytokine sets such as common cytokines, inflammatory cytokines, and chemokines.
Cytokine PCR arrays can be used for several different kinds of applications. In basic research, the array can be used to measure the response of cytokines to a stimulus, such as a candidate drug compound. The array can also be used to monitor biological samples such as those from human or animal, or cell culture samples. Patterns of expression of cytokines serve as phenotypic markers for certain biological processes.
A forthcoming product from SA Biosciences will expand the biological utility of the cytokine array by adding the functionality to identify transcription factors which regulate cytokines. Says Jeffrey Hung, PhD, Senior Director of Marketing at SA Biosciences, “Something we are going to launch pretty soon is a product we called 'Biology on Array' ...oftentimes people ask what transcription factor regulates my cytokine? Biology on Array is an innovative platform to quickly get an answer to that question.”
Cytokine analysis can also be carried out in a cell-based assay format. Hermann Bohnenkamp, PhD, a Product Manager for Miltenyi Biotec, has been tracking customer trends in the area of cytokine analysis. Miltenyi Biotec offers a cytokine secretion assay, which has the advantage that it can be used for the identification of live cytokine-secreting cells, in contrast to other methods in which the cells are destroyed. The assay uses fluorochrome-conjugated anti-cytokine antibodies. Cells of interest can also be enriched by MACS Technology, which is a column-based magnetic cell separation method. The enriched cells can then be easily cultured and used for downstream experiments.
Applications for cytokine secretion assays are very diverse, but the technology has been especially attractive to researchers carrying out adoptive T cell transfer in animal models. Cancer, and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis are especially amenable to analysis by cytokine secretion assay. Says Bohnenkamp, "One important thing is to include appropriate controls in the cytokine analysis experiment. Lots of people are using activation markers in the case of CD4 T cells. We think that CD154 is currently the best marker for this purpose regarding background and specificity. Therefore, we have also developed a variety of antibodies to monitor CD154 upregulation by flow cytometry."
In addition to being an object of inquiry, cytokines can also become research tools or even therapeutics. Symansis produces cytokines for the research market, emphasizing proteins produced in human cells with authentic post-translational modifications. They acquired their cytokine business from Apollo Cytokine Research in Sydney, Australia, and transferred the manufacturing method over a thousand miles to their headquarters in Timaru. After doing full validations on all of their products, they are up and running with their new catalog of one hundred and twenty cytokines.
Proteins for research are commonly synthesized in E. coli or chinese hamster ovary (CHO) cell lines. These proteins have all of the right amino acids, but they lack the correct post-translational modifications. Says Peter Foster, PhD, CEO of Symansis, “One of the problems we had early on was that people would say a human protein is a human protein. They didn't realize the significance of producing the human protein in a human cell.” The resulting proteins, according to Foster, are more biologically active and have a longer half life.
An example is erythropoietin (EPO), which is a highly glycosylated protein. EPO regulates the proliferation and differentiation of hematopoietic stem cells. In culture, cells grown with human-expressed EPO exceeded CHO-expressed EPO in morphological maturity and expressed more hemoglobin.
The study of cytokines has great potential to expand scientific knowledge of biological systems, especially in the area of biomarker discovery. Panels of biomarker proteins, such as cytokines, can be used not only for diagnostic purposes, but to monitor response to therapy, to predict the course of disease, or as a companion diagnostic in drug development. However, some significant challenges exist in the study of cytokines and other proteins in human tissues. The range of abundance of clinically relevant proteins spans twelve orders of magnitude, and some cytokines are on the extreme low end of this range. This means that at the current time, no single platform technology can analyze all of the cytokines in blood or serum. Instead, scientists rely on platform technologies like cytokine array, cytokine functional assays, cytokine secretion assays, and real-time PCR to analyze samples that are enriched for the protein targets.
In addition, scientists are realizing the significance of human expression of cytokines. Using cytokines with all of the proper post-translational modifications increases the likelihood of a successful outcome of a study. As more refined understanding of cytokine functions within biological networks is achieved, it will be increasingly important to use recombinant proteins that accurately represent these functions.