Cytokines: from technology to therapeutics.

Cytokines are playing an ever-increasing role in the treatment of human disease. The characterization of these proteins plays a vital role in their development as useful therapeutic agents. Physicochemical techniques can produce information about the structure and composition of cytokine therapeutics but cannot yet predict their biological activity, for which biological assays are required. Because of the large number of techniques available and the variety of products requiring analysis, the tests used to characterize cytokine products must be both appropriate for the product and adequately controlled if the information they provide is to be of value.

Detection and measurement of cytokines.

Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.

The binding of ligands to the 55 kDa component of the interleukin-2 receptor triggers increased turnover of phosphate bound to an 85 kDa protein. Evidence for the role of cyclic AMP.

The cell-surface receptor for interleukin-2 (IL-2) consists of two unlinked polypeptides of 55 and 75 kDa (p55, p75). The monoclonal antibody antiTac binds to p55 alone. We show here that the binding of either IL-2 or antiTac to the surface of T lymphocytes triggered the generation of cAMP. Reagents which activate adenyl cyclase by stimulation of its guanine nucleotide-binding protein (Gs) also stimulated increases in cAMP. All of the above reagents, and cAMP itself, stimulated the turnover of phosphate residues bound to serine and threonine residues of an 85 kDa protein. The data provide evidence that the binding of ligands to the p55 component of the IL-2 receptor generates a biochemical signal by the stimulation of adenyl cyclase via Gs, and that the consequent generation of cAMP and activation of cAMP-dependent protein kinase modulates the turnover of p85-bound phosphate groups.

Interleukin-2 binding to activated human T lymphocytes triggers generation of cyclic AMP but not of inositol phosphates.

Human T lymphocytes stimulated with phytohaemagglutinin undergo a single round of cell division. Further proliferation is dependent on the lymphokine interleukin-2 (IL2) [(1987) Immunology 60, 7-12]. We show here that binding of IL2 to its receptors on the lymphocyte surface triggers the generation of cyclic AMP. In contrast, generation of inositol phosphates from the breakdown of inositol lipids was not detected. We suggest that cyclic AMP may play a role in the transduction of the IL2 proliferative signal in T lymphocytes.

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin.

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.

The biological properties, assay, and standardization of interferon-alpha: a need for a WHO collaborative study.

Interferon-alpha (IFN-alpha) exists as a range of closely related, biologically active proteins and has been the subject of extensive research and clinical investigation. Standardization of IFN-alpha and the uniform reporting of IFN-alpha activity in International Units (IU) is critical to preclinical research and the clinical development of IFN-alpha products as therapeutic agents. Currently, several different IFN-alpha-containing reference preparations, established as World Health Organization (WHO) International Standards (IS) for particular IFN-alpha proteins (mixtures or single molecular species) are available for assay calibration. Nevertheless, the heterogeneous nature of IFN-alpha has raised standardization issues relating to the activity of individual IFN-alpha proteins, hence-forth termed subtypes, in the various biologic assays used for determining IFN-alpha levels. These issues include the question of parallelism of dose-response curves among particular IFN-alpha subtypes and different, naturally produced IFN-alpha subtype mixtures, for example, leukocyte IFN-alpha, and the applicability of IU of IFN-alpha activity defined by antiviral assays to alternative biologic assays, for example, antiproliferative assays. To address such issues, a WHO Consultative Group on Cytokine Standardization requested that the National Institute for Biological Standards and Control (NIBSC) and the Centre for Biologics Evaluation and Research (CBER) organize an international collaborative study to compare the activities and relative potencies of the several available IFN-alpha preparations, including those derived from human cells containing mixtures of IFN-alpha subtypes and those derived by rDNA methods containing single IFN-alpha subtypes, in different assays. To date, 111 participants in 32 countries have been recruited to the study and have agreed to assay a total of 17 different natural and recombinant IFN-alpha preparations or a defined subset thereof in specific in-house assays. The assay results generated will be statistically analyzed and evaluated to address the stated issues and to assess whether any individual IFN-alpha preparation is suitable to serve as an IS for all IFN-alpha preparations or whether more than one IS will be needed for this purpose.

Laboratory protocols for the quantitation of cytokines by bioassay using cytokine responsive cell lines.

Cytokines have been shown to be involved in many physiological processes, including the maintenance and control of a competent haematopoietic/immune system. Abnormal cytokine secretion or synthesis can cause a wide range of pathological disorders. Cytokines have also been shown to have therapeutic potential in many diseases. In order for the role of cytokines to be evaluated in normal and pathogenic processes, it is vital that appropriate assay systems are used to measure their levels in different biological fluids. Whilst immunoassays maybe a more convenient method for quantitating cytokines, they only measure immunologically reactive material. They may or may not detect biologically inactive material, such as cytokines bound to soluble receptors or degraded cytokine molecules. Bioassays, however, detect biologically active cytokines and can be as accurate and precise as immunoassays. The purpose of these protocols is to provide practical stepwise methods for the bioassay of cytokines using cytokine responsive cell lines. They include tables of the most useful currently available cell lines for the detection of cytokines and how to maintain them. In addition, these protocols provide all the materials and methods in a logical step-by-step procedure to carry out bioassays. Information is provided on possible pitfalls and general problems associated with bioassays and how to overcome them. These protocols should be valuable to scientists new to the cytokine field as well as experienced scientists who require a consensus methodology and access to information on cell lines useful for cytokine bioassays.

Quantitative cell line based bioassays for human cytokines.

Research in cytokine biology is ever increasing and it is clear that cytokines are involved in a wide range of pathological and physiological processes. The validity of such research relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Amongst the currently available methods for measuring cytokine levels, it is only the biological assay of samples that can directly provide estimates of biologically active cytokines present in test samples. Of the several bioassay systems available for detecting cytokines, cell line based bioassays are the easiest to perform and provide the most precise and accurate data. The suitability of any cell line for bioassaying a particular cytokine depends on several criteria such as sensitivity, ease of growth maintenance, and cytokine specificity. The design and analysis of cell line bioassays is also important in providing valid estimates of cytokine levels. We review the most useful cell lines currently available for bioassaying cytokines and discuss the design advantages and limitations of cytokine bioassays.

The international standard for granulocyte-macrophage colony stimulating factor (GM-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Two ampouled preparations of granulocyte-macrophage colony stimulating factor (GM-CSF) have been evaluated by twenty nine laboratories in eleven countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/646) was established as the international standard for GM-CSF.

A novel, sensitive bioassay for transforming growth factor beta.

We have developed a simple, sensitive bioassay for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) based on the ability of these cytokines to inhibit the interleukin-5 induced proliferation of the erythroleukaemia cell line, TF-1. This assay is rapid, reproducible and sensitive to less than 500 fg/ml of TGF-beta 1, and 5-10 pg/ml TGF-beta 2. The assay is 100-1000-fold less sensitive to other inhibitory molecules such as interferon-beta, interferon-gamma and TNF-alpha. The assay can be made specific for TGF-beta 1 or TGF-beta 2 by including specific neutralising antibodies for TGF-beta 1 or TGF-beta 2. The assay can recognise all the readily available recombinant molecular species of these molecules as well as the natural proteins produced from human and bovine platelets and detects TGF-beta in serum samples.

Immunoassays for detecting cytokines: what are they really measuring?

In summary, many factors influence the estimates of cytokine levels provided by immunoassays. If immunoassays are to supply data which are valid and useful to researchers and clinicians, these factors must be fully investigated during assay design and construction.

The international standard for granulocyte colony stimulating factor (G-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Three ampouled preparations of granulocyte colony stimulating factor (G-CSF) have been evaluated by 29 laboratories in 11 countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/502) was established as the international standard for G-CSF.

The international standard for macrophage colony stimulating factor (M-CSF). Evaluation in an international collaborative study.

Three ampouled preparations of macrophage colony stimulating factor (M-CSF) were evaluated by 23 laboratories in nine countries for their suitability to serve as international standards for this material. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), the preparation in ampoules coded 89/512 was established as the international standard for M-CSF.

Implications for the assay and biological activity of interleukin-8: results of a WHO international collaborative study.

Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-8 can have different biological specific activities, even though all were produced using E. coli. It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays. In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.

WHO cytokine standardization: facilitating the development of cytokines in research, diagnosis and as therapeutic agents.

The development and widespread application of recombinant DNA technology has dramatically increased the number of cytokines available for clinical evaluation. New and novel cytokines are being discovered, cloned and entered into clinical trials at such a rate that it is often the case that the biological activities of these proteins are poorly understood during their development as therapeutic agents. In addition, manufacturers of any one cytokine can produce the protein from different cellular sources resulting in materials that exhibit markedly different specific activities. When estimating the amount of biological activity of different preparations with different specific activities by bioassay, mass units cannot be used and biological activity is therefore expressed as 'biological potency units'. The biological unit requires definition by a standard that is assay-independent (especially when measuring a particular type of biological activity). In many cases, a variety of assay methods will be available and the material chosen for a standard should ideally be suitable for use with as many of them as possible. Once the unit is defined, this can be used in any laboratory, thus providing a means of ensuring uniformity throughout the world in the designation of potency of different biological preparations. The World Health Organisation (WHO) standardization programme involves the production of biologically stable, well characterised potency and immunoassay standards that are available world-wide using a single international unitage. Over the years, WHO international standards have been used to dramatically reduce the variation in estimates of cytokine preparations within and between laboratories for immunoassays and bioassays. WHO international standards are primary reference preparations against which secondary, or working standards (including regional standards, national standards, pharmacopoeial standards and in-house working standards) can be calibrated.

Implications for the assay and biological properties of interleukin-3. Results of a WHO international collaborative study.

Five preparations of interleukin-3 (IL-3) have been evaluated by 28 laboratories in 12 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for IL-3 and interleukin-4 (IL-4). The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the biological assays contributed to this study that different recombinant preparations of IL-3 can have very different biological specific activities, including those from the same source (i.e., E. coli). Biological assays of IL-3 were significantly more consistent in their estimates of levels of IL-3 than the immunoassays, suggesting an unusual pattern of epitope recognition amongst the antibodies included in the immunoassays. This study also illustrates the point that the level of cytokine measured by immunoassay does not necessarily reflect the biological potency of the cytokine. On the basis of results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-3 (91/510) was established as the international standard for interleukin-3 with an assigned unitage of 1700 IU/ampoule.

Implications for the assay and biological activity of interleukin-4. Results of a WHO international collaborative study.

Five ampouled preparations of interleukin-4 (IL-4) have been evaluated by 36 laboratories in 14 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for interleukin-3 (IL-3) and IL-4. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-4 can have very different biological specific activities, including those from the same source (i.e., E. coli). In addition, immunoassay estimates of IL-4 levels did not correlate with those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. It is of interest that the estimates provided by the different bioassays were less variable than those produced by the immunoassays, suggesting that bioassays can be as accurate, if not more so, than immunoassays. The large reduction in the variability of estimates with the inclusion of a single reference preparation clearly illustrates the need for a single standard to assay IL-4. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-4 (88/656) was established as the international standard for interleukin-4 with an assigned unitage of 1000 IU/ampoule.

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7.

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg/ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.

An antiproliferative bioassay for interleukin-4.

Interleukin-4 (IL-4) is currently being used for therapeutic intervention in a wide range of malignant diseases as an antitumour agent. Although bioassays have been developed that measure the proliferative capacity of IL-4, none measure the antiproliferative activity of this molecule. We have developed a simple, sensitive bioassay for human IL-4 based on the ability of this cytokine to inhibit the proliferation of the human lung carcinoma line, CCL-185, an easy to maintain, cytokine independent, cell line. It is rapid, reproducible and sensitive, able to detect 2 pg/ml IL-4. The assay is completely unresponsive to all other interleukins from IL-2 to IL-12, to the colony stimulating factors and transforming growth factor beta and is 100-fold less sensitive to interferon-alpha, tumour necrosis factor-alpha, IL-1 beta and IL-13. The assay can be made completely specific for IL-4 by including specific neutralizing antibodies for IL-4 and is suitable for the estimation of IL-4 in both plasma and serum samples.

An anti-cytokine bioactivity assay for interferons-alpha, -beta and -omega.

Interferons-alpha and -beta (IFN-alpha and -beta) are cytokines that are widely known to induce potent anti-viral activity. However, it has become increasingly apparent that IFN-alpha and -beta exert a variety of other biological effects, including anti-tumour and immunomodulatory activities and are increasingly used clinically to treat a range of malignancies, myelodysplasias and autoimmune diseases, e.g., IFN-beta for multiple sclerosis. The most widely used bioassays for the IFNs are based on their anti-viral activity, but these do not predict the biological activity of the IFNs in anti-tumour and immunomodulatory therapies. Thus, we have developed anti-cytokine-based bioassays that may be more reflective of such activity and which have several advantages over existing anti-viral bioassays. The anti-cytokine bioassay is based on the ability of IFN-alpha, -beta and -omega to inhibit granulocyte-macrophage-colony-stimulating factor (GM-CSF) induced proliferation of the erythroleukaemic cell line TF-1. This assay can take only 24 h, is sensitive to 200 fg (0.04 IU) IFN-alpha or -beta and 100 fg (0.02 IU) IFN-omega and is able to detect down to these levels in serum or plasma samples. The usefulness of anti-cytokine bioassays for IFN-alpha, -beta and -omega is not restricted to the GM-CSF/TF-1 cell format and other alternatives are available, such as erythropoietin (EPO)/TF-1 cells and EPO/UT-7-EPO cells. These assays can be made specific for each of the IFNs by including neutralising antibodies in the bioassay.