Bioassays for cytokines

10.1 Bioassays for cytokines

A vast array of published information is available on the immunoregulatory molecules known as cytokines that are secreted by cells and thus modulate the behaviour, function or differentiation state of other cells.

With the original nomenclature, a cytokine produced by lymphocytes was called a lym- phokine, and a cytokine produced by the monocyte lineage of cells, a monokine. However, such terms are restrictive and misleading; for example, the so-called lymphokine, interleukin 6, is produced by more than one cell type in addition to leucocytes: fibroblasts, epithelial cells, keratinocytes, monocytes, microglial cells, endothelial cells and uterine stromal cells.

Many cytokines that are intimately involved in the regulation of specific and non-specific immune function (e.g. as illustrated for IL-1 and IL-2 in Fig. 10.1) probably pre-date adaptive immunity in evolution and have retained important (more important?) roles outside the immune system. The cytokines so far identified have been shown to interact with their target cell via a specific receptor, though the route(s) of signal transduction to the nucleus remain to be defined.

Recombinant DNA technology, protein sequence analysis, the use of monoclonal antibodies and the development of a variety of bioassays has enabled many of the properties of these cytokines to be demonstrated. The precise physiological role of a cytokine is often difficult to determine; many have been shown to have several activities in vitro and so it is difficult to pre- dict which activity might have in vivo relevance. Several cytokines have been shown to have

10.1BIOASSAYS FOR CYTOKINES

Quiescent T Antigen presented

in combination lymphocyte with MHC gene

Antigen

T lymphocyte

product

Antigen receptor

Activated T lymphoblast

IL-2 receptor

Secretion of IL-2— autostimulation and stimulation of other activated cells

IL-2

Antigen-independent; growth factor (IL-2) driven proliferation

IL-2 receptor down-regulates

Clone of quiescent T h lymphocytes

Fig. 10.1 Regulation of antigen-specific clonal proliferation of T lymphocytes by cytokines. Antigen bridging between the antigen-presenting cell (APC) and T lymphocyte results in secretion of IL-1 by the APC. Interleukin 1 and antigen activate the T lymphocyte to express its preprogrammed effector function, in this case secretion of IL-2. At the same time, however, the T lymphocyte also up-regulates its high-affinity IL-2 receptors and so can respond to extracellular IL-2, including its own. Whereas the initial activation phase is antigen driven, the later proliferative phase is largely in response to IL-2. Feedback control is achieved in turn by the down-regulation of the IL-2 receptor by IL-2 in the presence of diminishing antigen concentration.

convergent effects in the immune system, e.g. both IL-2 and IL-4 maintain the proliferation of activated T cells. Although the molecules are structurally different, a response to a cytokine is elicited following cytokine binding to the specific receptor. The nature of the response is depend- ent on the type of cell being stimulated. When deciding on a method to measure cytokines there are two key assay systems:

1 bioassay that measures biological activity, and

2 immunoassay, such as an ELISA, that measures the concentration of the cytokine within the body fluid/cell lysate of interest, but does not determine whether it has any activity. It seems likely that synergy or inhibition of activity between cytokines may occur, thus pro-

viding opportunities for highly discriminatory fine-tuning of the immune system. Nomenclature of the cytokines is confusing as many cytokines have been isolated independently in a number of different laboratories throughout the world. Thus similar or identical molecules are known by

a variety of names. It is possible to standardize assays via a growing collection of international and national standard cytokine preparations obtainable from the National Cancer Institute (Bethesda, USA), or the National Institute for Biological Standards and Control (South Mimms, UK).

The majority of the cytokines described here are available as recombinant proteins, derived from in vitro expression in prokaryotic or eukaryotic systems. Although most recombinant molecules have a comparable activity they are not identical to the native cytokine, especially if derived by prokaryotic expression. In particular, glycosylation patterns may vary quite considerably.

Most cytokines occur in similar or equivalent forms throughout the mammals. However, although nucleotide and amino acid sequence analysis shows considerable homology between the species for some cytokines, the cross-reactivity between species is low or unidirectional. For example, human IL-2 will stimulate lymphocytes from the rat and the mouse, but rodent IL-2 cannot stimulate human lymphocytes (see the useful Table prepared by Horst Ibelgaufts, University of Munich, at the aptly named website: http://www.copewithcytokines.de/cope.cgi?001174).

To ensure that a functionally active cytokine is present in an unknown sample, a bioassay is required. Monoclonal antibodies have been developed into commercially available enzyme- linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) kits. Dot blotting or northern analysis with DNA probes can also be used to define whether a cell is capable of cytokine produc- tion, though this is not as sensitive as a bioassay and not quantitative to any degree of accuracy.

Calculation of results National/international standards for some cytokines are available. Here are some recommenda-

tions for when considering undertaking cytokines assay studies.

1 Calibrate your own laboratory standard which can then be used as a reference for all further assays.

2 Make a bulk preparation of the cytokine of interest, aliquot into convenient samples for storage at –70°C and do three or four repeat assays on aliquots of this stored material.

3 The standard preparation should give reproducible results (albeit some variation due to het- erogeneity in the responder-cell population), and a mean of the dose–response curves will enable you to identify the dilution of standard which gives 50% stimulation of activity, cell killing, etc. Assign an arbitrary number of units to this and use as a positive control in all future experiments.

10.1BIOASSAYS FOR CYTOKINES

4 The cross-reactivity between species is variable, so care must be taken when attempting to determine cytokine activity on cells from different species. Cytokines are molecules with a defined structure and with potent effector functions on cells.

For simplicity, immunoassays, either radio- or enzymic, should be ideal for estimating cytokines. Antibodies provide very precise recognition of molecular structure, especially in two-site assays where two monoclonal antibodies recognize distinct epitopes on the cytokine, but such assays frequently fail to give correct information on the activity of the cytokine. With good standards they should give reliable information on mass.

Failure of correlation between recombinant cytokine mass and activity can be due to a num- ber of causes. Glycosylation to produce the mature protein can be an important characteristic that has a functional role in a specific cytokine. However, if that cytokine is produced as a recom- binant protein, e.g. in yeast, mammalian oligosaccharides will not be added and this can affect the biological activity. Even cytokines produced by cultured mammalian cells will often have altered glycosylation unless extreme care is taken to maintain nutrients and culture conditions in an optimal steady state. In vivo soluble cytokine receptors are often produced, frequently in greater quantity than the cytokine, and these may inhibit function while the cytokine is still detected in an immunoassay. For the above reasons bioassays are generally preferred when stud- ies are being made that relate to cytokine function.

Problems with bioassays Specificity is a key issue; cytokines are usually assayed in vitro on populations of cells and most

cells have receptors for more than one cytokine so that care must be taken to ensure that the correct cytokine is being detected. To overcome this it may be advisable to combine bioassay with the specificity of antibody. If a neutralizing anticytokine antibody is available, the assay may

be performed both with and without the antibody, the difference in activity being due to the cytokine in question, and it is essential to be aware that this still cannot prevent errors caused by cytokines yet to be discovered which may well be present in the test samples.

Cytokines may be assayed in whole animals, cells isolated from animals or on cell lines or clones. Obviously whole animal experiments are only ethical where no other method is available and their use is generally restricted to the later stages of drug development, for instance for testing the in vivo anticancer effects of a cytokine. An alternative to using a live animal would be to take cells from particular organs, such as spleen cells or thymocytes, and examine the effects of cytokines on these particular mixtures. This gives a good idea of how the cytokine relates to the in vivo mix of cells, but is highly complex for simply assaying cytokine activity. Sometimes, how- ever, it is difficult to get hold of the correct cell line and we show below how to use animal- derived cell preparations and give recommendations for individual cell lines. If a large number of assays are to be performed over a period of time cell lines should be used as this will cut down on the requirement to use animals and will provide greater consistency than using cells from differ- ent animals each time.

Sample collection Cytokines are frequently measured in blood samples and great care is needed to avoid artefacts.

Lithium heparin, in certain collection tubes, is contaminated with endotoxin which will have

C H A P T E R 1 0: The cytokines C H A P T E R 1 0: The cytokines