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Methods for analyzing cytokines
Methods 38 (2006) 235–236 www.elsevier.com/locate/ymeth
Introduction
Methods for analyzing cytokines
Cytokines are small peptides that modulate several important aspects of the inflammatory system. They are part of a normal physiologic process and also part of the host response to injury and stimulation. Cytokines are a multibillion-dollar industry when one considers that cytokine inhibitors are used for the treatment of chronic inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Cytokines exhibit a wide range of activities ranging from the activation of immune cells to the regulation of body temperature. They have the potential to act in an autocrine manner when the cell that secreted the cytokine responds to that cytokine. An example of this would be when a neutrophil secretes IL-8 and then responds to this secreted IL-8. Paracrine actions are evident when the secreted cytokine acts on neighboring cells. This occurs when Kupfer cells in the liver secrete IL-6, and this secreted IL-6 then induces neighboring hepatocytes to synthesize acute phase proteins. Finally, endocrine effects may occur when the cytokine travels to a distant organ to exert its biological activity. A classic example of this occurs when erythropoietin, produced by the kidney, moves to the bone marrow to augment production of red blood cells. Given the wide range of activities and locations where cytokines may be observed, it follows that a wide range of analysis methods for detecting the cytokines in these different scenarios would be required. A technology that is appropriate for measuring cytokines in liquid phase, such as the circulating blood, may lack the sensitivity and specificity needed to detect the presence of the cytokine within tissues or individual cells. In this issue of Methods, we have solicited contributions from investigators that detail multiple technologies for the detection and quantification of these potent protein mediators. In the first article, Anthony Meager discusses the evaluation of the biological activity for cytokines. This is an important aspect that must be remembered in the analysis of cytokines, and one that is frequently not addressed. Virtually all modern technology for the cytokine assays are immunologically based and do not actually measure if the cytokine retains functional activity.
1046-2023/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2006.02.001
Because all cytokines are proteins, they begin life as messenger RNA that is translated into the protein. To precisely localize where cytokines are being produced within a particular organ, in situ hybridization for detection of mRNA has been used with great utility. In the second article, the Wellstein group carefully reviews this methodology. Once the mRNA has been translated, the cytokine is initially produced within the cytoplasm of the cell prior to being packaged and exported. A popular technology has developed where intracellular cytokines may be analyzed by flow cytometric techniques. This has been used with great success for analyzing cytokine production by T cells. In the third article, Gauduin describes the working nature of this methodology. Another technique for measuring individual cell cytokine production is the ELISASPOT assay. Cox and colleagues review the perils and pitfalls as well as the strengths of this technique. Western blot analysis is a classic, time-honored method for the detection of proteins. Because cytokines are proteins, they may be detected by Western blot analysis. The Western blot has the advantage of working in tissue homogenates, plasma, and the cell culture supernatants. Kurien and Scofield review this methodology in the fifth article in this issue. One of the mainstays for measuring cytokines in most laboratories is the ELISA. Most current ELISAs are a double antibody method, although other variations exist. There are multiple vendors that sell either prepackaged kits for measuring cytokines or individual components. In the sixth article, de Jager and Rijkers carefully review the basic concepts behind this robust methodology. In the seventh article, Osuchowski and Remick describe a variation of the traditional ELISA in which samples are sequentially measured in different cytokine assays. This has the advantage of being able to stretch a small volume of sample to measure multiple mediators. Bead array assays are becoming increasingly common for the measurement of several mediators, including cytokines. These assays are gaining prominence although they are still relatively novel. Two separate articles are devoted
236
Introduction / Methods 38 (2006) 235–236
to descriptions of the bead array assays. Hill and Martins describe the basic concepts, and Elshal and McCoy evaluate the performance of these newer assays. The aptamer technology permits detection of specific proteins by determining their binding to discrete fragments of nucleic acid. The tenth article in this issue is devoted to a discussion of the aptamer technology by Le and colleagues. Microdialysis sampling permits the in vivo detection of specific proteins. Ao and Stenken describe the methodology, including how it may be used for the delivery of cytokines as well as for their detection. Proteomics technology is being used for the discovery of new mediators, as well as quantification of previously described proteins. Boyle and colleagues describe a proteomics approach to cytokine measurement is described in the twelfth article in this issue.
Multiple modalities exist for the accurate and rapid and accurate quantification of cytokines. This issue contains well-written descriptions of the major methods that are currently available. The papers should serve as an excellent resource for those scientists who are interested in measuring cytokines, as well as those who desire a deeper understanding of the basic concepts of the techniques. Daniel G. Remick * Jill I. Granger Department of Pathology, University of Michigan Health System, 1301 Catherine Street, Ann Arbor, MI 48109-0602, USA E-mail address: [email protected]. Accepted 2 February 2006
*
Corresponding author. Fax: +1 734 763 6476.
Introduction
Methods for analyzing cytokines
Cytokines are small peptides that modulate several important aspects of the inflammatory system. They are part of a normal physiologic process and also part of the host response to injury and stimulation. Cytokines are a multibillion-dollar industry when one considers that cytokine inhibitors are used for the treatment of chronic inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Cytokines exhibit a wide range of activities ranging from the activation of immune cells to the regulation of body temperature. They have the potential to act in an autocrine manner when the cell that secreted the cytokine responds to that cytokine. An example of this would be when a neutrophil secretes IL-8 and then responds to this secreted IL-8. Paracrine actions are evident when the secreted cytokine acts on neighboring cells. This occurs when Kupfer cells in the liver secrete IL-6, and this secreted IL-6 then induces neighboring hepatocytes to synthesize acute phase proteins. Finally, endocrine effects may occur when the cytokine travels to a distant organ to exert its biological activity. A classic example of this occurs when erythropoietin, produced by the kidney, moves to the bone marrow to augment production of red blood cells. Given the wide range of activities and locations where cytokines may be observed, it follows that a wide range of analysis methods for detecting the cytokines in these different scenarios would be required. A technology that is appropriate for measuring cytokines in liquid phase, such as the circulating blood, may lack the sensitivity and specificity needed to detect the presence of the cytokine within tissues or individual cells. In this issue of Methods, we have solicited contributions from investigators that detail multiple technologies for the detection and quantification of these potent protein mediators. In the first article, Anthony Meager discusses the evaluation of the biological activity for cytokines. This is an important aspect that must be remembered in the analysis of cytokines, and one that is frequently not addressed. Virtually all modern technology for the cytokine assays are immunologically based and do not actually measure if the cytokine retains functional activity.
1046-2023/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2006.02.001
Because all cytokines are proteins, they begin life as messenger RNA that is translated into the protein. To precisely localize where cytokines are being produced within a particular organ, in situ hybridization for detection of mRNA has been used with great utility. In the second article, the Wellstein group carefully reviews this methodology. Once the mRNA has been translated, the cytokine is initially produced within the cytoplasm of the cell prior to being packaged and exported. A popular technology has developed where intracellular cytokines may be analyzed by flow cytometric techniques. This has been used with great success for analyzing cytokine production by T cells. In the third article, Gauduin describes the working nature of this methodology. Another technique for measuring individual cell cytokine production is the ELISASPOT assay. Cox and colleagues review the perils and pitfalls as well as the strengths of this technique. Western blot analysis is a classic, time-honored method for the detection of proteins. Because cytokines are proteins, they may be detected by Western blot analysis. The Western blot has the advantage of working in tissue homogenates, plasma, and the cell culture supernatants. Kurien and Scofield review this methodology in the fifth article in this issue. One of the mainstays for measuring cytokines in most laboratories is the ELISA. Most current ELISAs are a double antibody method, although other variations exist. There are multiple vendors that sell either prepackaged kits for measuring cytokines or individual components. In the sixth article, de Jager and Rijkers carefully review the basic concepts behind this robust methodology. In the seventh article, Osuchowski and Remick describe a variation of the traditional ELISA in which samples are sequentially measured in different cytokine assays. This has the advantage of being able to stretch a small volume of sample to measure multiple mediators. Bead array assays are becoming increasingly common for the measurement of several mediators, including cytokines. These assays are gaining prominence although they are still relatively novel. Two separate articles are devoted
236
Introduction / Methods 38 (2006) 235–236
to descriptions of the bead array assays. Hill and Martins describe the basic concepts, and Elshal and McCoy evaluate the performance of these newer assays. The aptamer technology permits detection of specific proteins by determining their binding to discrete fragments of nucleic acid. The tenth article in this issue is devoted to a discussion of the aptamer technology by Le and colleagues. Microdialysis sampling permits the in vivo detection of specific proteins. Ao and Stenken describe the methodology, including how it may be used for the delivery of cytokines as well as for their detection. Proteomics technology is being used for the discovery of new mediators, as well as quantification of previously described proteins. Boyle and colleagues describe a proteomics approach to cytokine measurement is described in the twelfth article in this issue.
Multiple modalities exist for the accurate and rapid and accurate quantification of cytokines. This issue contains well-written descriptions of the major methods that are currently available. The papers should serve as an excellent resource for those scientists who are interested in measuring cytokines, as well as those who desire a deeper understanding of the basic concepts of the techniques. Daniel G. Remick * Jill I. Granger Department of Pathology, University of Michigan Health System, 1301 Catherine Street, Ann Arbor, MI 48109-0602, USA E-mail address: [email protected]. Accepted 2 February 2006
*
Corresponding author. Fax: +1 734 763 6476.