Minireview DETECTING CYTOKINE PRODUCTION AT THE SINGLE-CELL LEVEL Claire E. Lewis Cytokines are versatile mediators of intercellular communication. Their functional diversity has aroused considerable interest and prompted the rapid development of a number of techniques for their detection and measurement. However, conventional cytokine assays measure only their bulk release by large numbers of cells and give no indication of the identity or frequency of producer cells. Here, the advantages and disadvantages of a relatively new approach to detect cytokine production by single cells are reviewed. Copyright o 1991 by W.B. Saunders Company

Since the advent of recombinant DNA techniques and their application to cytokine biology, considerable advances have been made in the methods by which these soluble protein mediators are detected. Fundamental problems remain, however, in the analysis of cytokine production. Cytokines are often inducible proteins which are only transiently stored. They also have overlapping biological actions and can be released or coexist with carrier proteins or competitive inhibitors. This has prompted careful attention to the selection of appropriate and meaningful techniques for their accurate measurement. A wide range of bioassays were initially developed to measure cytokine levels on the basis of their bioactivity in vitro (for review, see reference 1). Although the application of recombinant cytokine products and specific cytokine antibodies has helped define the specificity of some bioassays, these assays are often time-consuming and cumbersome. Their use has also led to problems of specificity, since they rarely involve the effect of a single cytokine on a single cell type. Cytokines are probably not released in isolation, but rather form part of a heterogeneous mixture of enzymes, serum proteins, hormones, and other cytokines. This can lead to ambiguous results that are difficult to interpret. Alternative methods for measuring the net amount of cytokine secreted into culture supernatants or body fluids by large numbers of cells include radioimmunoassays and enzyme-linked immunoassays.‘,’ However,

Nuffield Department of Pathology Oxford, John Radcliffe Hospital, Copyright 0 1991 by W.B. Saunders 1043-4666/91/0303-0013$05.00/0 KEY single

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WORDS: cell/cellular

cytokines/cytokine level/assay

and Bacteriology, University Oxford OX3 lUN, UK. Company productionicytokine

release/

of

even these forms of cytokine measurement can only estimate the secretory activity of the entire cell population. The use of such bulk-release detection methods often assumes that, under any one condition, cells of a given phenotype show identical or similar levels of cytokine-secreting behavior. Further, the sensitivity of these techniques can be limited in situations where relatively few producer cells are present, or release sub-threshold levels of cytokine. It may also be difficult to ensure the complete elimination of contaminating cell types which secrete the same cytokine and/or a number of cytokines with overlapping or antagonistic activities. These important limitations, together with recent reports of potential plasticity in both the frequency of cytokine-producing cells314and the amount of cytokine released per ce11,5*6 have prompted the rapid evolution of various strategies for studying cytokine production at the single-cell level (see Tables 1 and 2).

DETECTION OF CYTOKINE GENE PRODUCTS IN INDIVIDUAL CELLS: mRNA AND INTRACELLULAR PROTEIN Detection of mRh!A Transcripts Table 1 indicates the range of techniques currently being applied to detect cytokine-specific mRNA molecules at the cellular level. Both isotopic and non-isotopic labeling methods of in situ hybridiization have been used to detect cytokine mRNA in tissue sections or cell preparations. In situ hybridization is an extremely useful technique, in that (1) the rapid emergence of different detection systems now permits the simultaneous localization of mRNA for more than one cytokine per cell, and (2) it can be coupled with conventional immunocytochemical methCYTOKINE,

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Detecting cytokine production Table 1, Techniques current list.

for detecting

the production

of cytokine mRNA transcripts

and intracellular

protein

Cytokine

Species of producer cell

IL-lp, IL-6, TNF-(r IL-2, IL-4, IFN-?/ IFN-B

Human Human Human

6 25 26

Polymerase chain reaction @‘CR)

IL-2

Human

8

Chimeric gene analysis

IL-2

Mouse

7

Could be combined with immuno-labeling methods to identify producer cell type(s) or the coincidental production of cytokine protein in cells expressing a cytokine gene.

TNF IL-1p IL-2 IL-6

Human Human Human Human

27 19 28 29 13 19

Rapid. Identifies producer cell w(s).

IL-la, p TNF-(Y, IL-6 TNF-B IFN-y, IL-2, IL-4 TNF IL-1p

Human Human Human

30 31 32 10 11 12

As above. Simultaneous production of more than one cytokine can be visualized.

Technique

(a) mRNA In situ hybridization (isotopic) (non-isotopic)

(b) Cytokine protein Immunocytochemistry

Immunofluorescence

Immuno-electron microscopy

Table 2.

Techniques

Technique

employed

in the detection Cytokine

Limiting dilution analysis

Reverse hemolytic plaque assay

Mouse Human

Reference

Advantages

Species of producer cell

Reference

Human

IFN-y IL-l, 2,6, IFN-y, GM-CSF TGF-b,

Human Human

33 34

Human

35

Cell blotting

IL-1 IL-2 IFN-y

Human Human Human

21 22 19

Elispot

IFN-y IFN-?/, TNF-a TNF, IFN-y

Human Human Mouse

IL-5, IFN-?/

Mouse

2 24 3 36 37

by single cells-the

Disadvantages

Can be combined with other techniques to identify producer cell type(s). Detection does not rely on presence of threshold number of producer cells. Non-isotopic method is rapid. Semi-quantitative. Greater sensitivity-detects lower numbers of transcripts.

As above. Highlights subcellular events involved in cytokine synthesis.

of cytokine release by single cells-the

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current

Relatively difficult technique. Contamination by other RNA/DNA molecules can be a problem. Non-quantitative. Relatively difficult technique. Time consuming.

Non-quantitative. Non-specific staining can be a problem. Cells responding to a given cytokine (by uptake) can be labeled as producer cells. Detects only antigenic, not necessarily bioactive, cytokine molecules. As above.

As above. Processing can destroy antigenicity of cytokine molecules.

list.

Advantages

Accurate. Quantitative. Enumeration of producer cells in a given population. Semi-quantitative. Sensitive. Rapid. Can be adapted to detect the release of more than one cytokine per cell. Producer cells can be readily immunophenotyped after the assay, thereby circumventing the need for purification of cells. As above, but quantitative.

Enumeration of producer cells. Sensitive. Rapid.

Non-quantitative. Isotopic method can pose a safety hazard and can be time-consuming.

Disadvantages

Only used for derived cytokines. Expensive. Bulky and time-consuming. Detects only antigenic, not necessarily bioactive, cytokine molecules.

As above. Expensive image analysis setup needed for quantification of color intensity of blots. As for the RHPA, but difficult to adapt for the identification of producer cell type(s).

186 I Claire E. Lewis

ods to demonstrate the simultaneous presence of the translated cytokine product and/or the phenotype of the producer cell6 Alternatively, the expression of the interleukin-2 (IL-2) gene has been visualized and quantified at the single cell level using chimeric gene analysis. In this method, the 5’ regulatory sequences from the mouse IL-2 gene were fused to the coding region of the Escherichia coli P-galactosidase (lad) gene. When stably reintroduced into a T-cell hybridoma, the expression of the reporter gene product (@galactosidase), which is similar to that for the resident IL-2 gene, is readily visualized.’ Criticisms of these two methodological approaches to mRNA analysis include those of insensitivity when low numbers of copies of mRNA are present in cells and the fact that the amount of mRNA present per cell is difficult to quantity. A third line of investigation has exploited the sensitivity of the polymerase chain reaction (PCR) to detect cytokine transcripts in individual human cells. This involves reverse transcription of total cellular mRNA to produce cDNA, followed by enzymatic amplification by PCR of the cytokine-specific cDNA using appropriate primers.* Interestingly, Gilliland et a1.9have recently reported the use of competitive PCR amplification to quantify the small amount of mRNA for GM-CSF or IL-3 present in as few as 200 cells. This then raises the exciting possibility of not only detecting, but also quantifying, the mRNA content of single cells by this method in the near future.

Detectionof Intracellular Cytokine The use of a wide range of immunostaining techniques to localize intracellular cytokines at both the light and electron microscopic level has recently been reported in a number of studies (see Table 1). This offers the facility of identifying producer cells, visualizing the production of more than one cytokine by an individual cell,” and highlighting the subcellular organelles involved in cytokine synthesis.“812 It should also be noted, however, that definitive studies correlating the synthesis and intracellular storage of cytokines with their subsequent release have yet to be performed. Indeed, a recent study has indicated that the production of cytokine mRNA and protein may not be tightly linked.13 In addition, cytokine associated with a given cell may not have been syntheisized by that cell, but may represent an internalized product of another cell type. Thus, extrapolating from the presence of intracellular cytokine to the release of the translated product has often been somewhat dubious.

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DETECTION AND QUANTIFICATION OF CYTOKINE RELEASE BY INDIVIDUAL CELLS Rapid advances in assaying technology have recently provided the cytokine biologist with several methods for detecting the release of cytokines by individual cells (Table 2).

Limiting Dilution Analysis (LDA) Reports by Kelso14 and Vie and Miller” have outlined the adaptation of limiting dilution analysis (LDA) to estimate the frequency of cytokine-producing cells and amount of cytokine released per cell. In this method, T lymphocytes are cultured with mitogens in limiting dilutions. After 2 to 7 days of culture, the quantity of cytokine released into the supernatant is measured by a sensitive bioassay. Proliferation is inhibited by irradiation (up to 2,000 rad) of producer cells prior to culture. Although quantitative, this approach has some major drawbacks. To date, LDA has only been adapted to measure the cytokines released by T cells. It is also a time-consuming and cumbersome process, and relies heavily on the availability of a sensitive assay to measure the level of cytokine released into the supernatant by each single cell in culture.

ReverseHemolytic PluqueAssay (RHPA) The hemolytic plaque assay was established to detect and enumerate individual immunoglobulinsecreting B lymphocytes. The reverse modification, or RHPA, was originally described for the detection of antigen secretion. In this assay secretory cells form plaques (zones of hemolysis around secretory cells) when incubated in a monolayer with protein A-coated ovine erythrocytes in the presence of a specific antiserum and complement. Since the size of hemolytic plaques has been shown to be directly proportional to the amount of product secreted per ce11,‘7~18 this technique can be used to quantify both the amount secreted per single cell and the frequency of cytokinesecreting cells in a given population. Recent studies have indicated the level of sensitivity of this immunoassay to be as low as lo-” M of secreted product per single ce11.5’17 A major advantage of the RHPA is the ease with which producer cells can be identified by routine immunocytochemistry upon termination of the assay.“19 This has proved useful as (1) the secretory activity of various cell types in culture can be visualized and quantified without the need for time-consuming attempts to purify cells prior to assaying, and (2) quantitative differences in the amount of cytokine secreted by cells of the same and different immunophe-

Detecting cytokine production

notype can be investigated. Furthermore, the subcellular events involved in the secretory activity of individual producer cells can be investigated by combining the RHPA with such valuable techniques as immunoelectron microscopy,17220autoradiography, and in situ hybridization (Lewis and co-workers, unpublished work). The RHPA has also recently been modified to offer the facility of visualizing the release of more than one secreted product by an individual ce11.20 The RHPA is one of the most versatile single-cell cytokine assays currently available, but it also has a number of limitations. As with all immunoassays, it is the release of the immunogenic, rather than the bioactive form of the molecule that is measured. These two may not always be concordant. It is also possible that producer cells could be affected by the presence of polyclonal antibody during the brief incubation period of the assay, causing abnormal patterns of cytokine secretion. Cell types such as monocytes/macrophages, T lymphocytes, and NK cells all bear Fc receptors. Although there is no evidence that this occurs, recent studies have circumvented the possibility by modifying the assay to run in the absence of free antibody by the use of erythrocytes coated with antibody prior to the assay (Lewis and co-workers, unpublished work).

Cell Blot Assay Briefly, this technique employs a protein-binding membrane to capture and visualize cytokine secretion. This membrane is labeled by routine immunocytochemistry using a primary antibody directed against the cytokine and an enzyme-linked secondary antibody coupled to a substrate to produce a color reaction. The amount secreted by each cell is quantified using computerized image analysis of the size and color intensity of the cytokine blots.19,21,22 It should be noted that analysis of blots by a high-powered (expensive) image analysis system is necessary for this method to be fully quantitative. Also, the possible influence on producer cells of their attachment to a membrane during the assay has yet to be investigated.

ELISPOT Assay This technique is based on the ELISA-plaque assay described by Sedgewick and Holt23 for the detection of individual antibody-secreting B cells. In the reverse form of this assay, single cells are incubated on a solid surface pre-coated with a specific cytokine antibody, which then binds cytokine secreted during the assay. The cells are then washed away and bound cytokine detected using a second monoclonal antibody that recognizes a different epitope on the cytokine mo1ecule.2~‘9~24

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Although it is a fast and reliable method for estimating the frequency of cytokine-producing cells in culture, the ELISPOT assay is not readily adapted to immunophenotype cytokine-secreting cells, since these are usually washed away during the procedure. However, Czerkinsky and co-workers2 have reported some degree of success in this area using a modified, if somewhat cumbersome, form of the assay. Full quantitation of the amount of cytokine released per single cell in the ELISPOT assay has yet to be reported. Limitations on the interpretation of data are also imposed by the presence of a layer of antibody molecules (as is true for the RHPA, although in the latter case the antiserum is free in solution), to which producer cells are attached during the assay. Although the various methods reviewed here offer greater sensitivity and versatility than more conventional assays for the net release of a cytokine product by large numbers of cells, these single-cell assays, by definition, visualize the output of cells in isolation rather than in tandem with neighboring cells. This limiting feature should be borne in mind when interpreting results obtained in this way, since such contacts may prove important in the regulation of cytokine production. As mentioned earlier, these extremely sensitive immunoassays and immunostaining methods may also suffer from another important shortcoming. They only detect immunogenic cytokine molecules. This may not necessarily be correlated with the bioactivity of the cytokine under study. In future studies it may prove advantageous to combine single-cell analysis with some of the more specific forms of bioassay available, thereby making them complementary rather than alternative techniques. Such an approach could provide data on the activity of both the individual producer cells present and the cytokine released into the supernatant during any given single-cell assay. REFERENCES 1. Hamblin AS (1988) Lymphokines. IRL Press at the Oxford University Press, Oxford, UK. 2. Czerkinsky C, Andersson G, Ekre H-P, Nilsson, L-A, Klareskog, L, Ouchterlony 0 (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. J Immunol Methods 111:2936. 3. Hutchings PR, Cambridge G, Tite JP, Meager T, Cooke A (1989) The detection and enumeration of cytokine-secreting cells in mice and man and the clinical application of these assays.J Immunol Methods 120:1-8. 4. Elias JA, Chien P, Gustilo KM, Schreiber AD (1985) Differential interelukin-1 elaboration by density-defined human monocyte subpopulations. Blood 66:298-301. 5. Lewis CE, McCarthy SP, Lorenzen J, McGee JO’D (1989) Heterogeneity amongst human mononuclear phagocytes in their secretion of lysozyme, interleukin-1 and type p transforming growth factor. Eur J Immunol19:2037-2043.

188 / Claire E. Lewis 6. Ogilvie AD, Wood NC, Dickens E, Wojtacha D, Duff GW (1990) In situ hybridisation. Ann Rheum Dis 49:434-439. 7. Emilie D, Peuchmaur M, Barad M, Jouin H, Maillot M-C, Couez D, Nicolas J-F, Malisson B (1989) Visualising interleukin 2 gene expression at the single cell level. Eur J Immunol19:1619-1624. 8. Brenner CA, Tam AW, Nelson A, Engelman EG, Suzuki N, Fry KE, Larrick JW (1989) Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNA’s from small numbers of cells. Biotechnique 7:1096-2002. 9. Gilliland G, Perrin S, Blanchard K, Bunn F (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Nat1 Acad Sci USA 87:27252729. 10. Andersson U, Andersson J, Lindfors A, Wagner K, Moller G, Heusser CH (1990) Simultaneous production of interleukin 2, interelukin 4 and interferon-y by activated human lymphoctyes. Eur J Immunol20:1591-1596. 11. Liu C-C, Detmers PA, Jiang S, Young JD-E (1989) Identification and characterization of a membrane-bound cytotoxin of murine cytolytic lymphocytes that is related to tumour necrosis factodcachectin. Proc Nat1 Acad Sci USA 86:3286-3290. 12. Singer II, Scott S, Hall GL, Limjuco G, Chin J, Schmidt JA (1988) Interleukin 1B is localized in the cytoplasmic ground substance but is largely absent from the golgi apparatus and plasma membranes of stimulated human monocytes. J Exp Med 176:389407. 13. Feldmann M, Brennan FM, Chantry D, Haworth C, Turner M, Abney E, Buchan G, Barrett K, Barkley D, Chu A, Field M, Maini RN (1990) Cvtokine uroduction in the rheumatoid ioint: implications‘for treatment. Ann Rheum Dis 49:480-486. * 14. Kelso A (1986) An assay for colony stimulating factor (CSF) production by single T lymphocytes: estimation of the frequency of cells producing granulocyte-macrophage CSF and multilineage CSF within a T lymphocyte clone. J Immunol136:2930-2937. 15. Vie H, Miller RA (1986) Estimation by limiting dilution analysis of human IL-2 secreting T cells: detection of IL-2 produced by single lymphokine-secreting cells. J Immunol 136:3292-3297. 16. Molinaro GA, Dray S (1974) Antibody-coated erythrocytes as a manifold probe for antigens. Nature 248:515-520. 17. Neil1 JD, Smith PF, Luque EH, Munoz de Toro M, Nagy G, Mulchahey JJ (1987) Detection and measurement of hormone secretion from individual pituitary cells. Recent Prog Horm Res 43:175-199. 18. Ahaerts W, Wouters A, Van der Massen D, Persoons A, Denef C (1988) A diffusion-adsorption model for the computation of the amount of hormone around a secreting cell, detected by the reverse hemolytic plaque assay. J Theor Biol 131:441-459. 19. Lewis CE (in press) Detection of cytokine production by individual cells. In Balkwill FR (ed) Cytokines: A Practical Approach, IRL Press at Oxford University Press, Oxford, UK. 20. Smith PF, Luque EH, Neil1 JD (1986) Detection and measurement of secretion from individual neuroendocrine cells using a reverse haemolytic plaque assay. Methods Enzymol 124:443454. 21. Gaffney EV, Stoner CR, Lingenfelter SE, Wagner LA (1988) Demonstration of IL-l (Y and B secretion by the monocytic leukemia cell line, THP-1. J Immunol Methods 122:211-218.

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22. Viselli SM, Mastro AM (1989) Detection and quantitation of IL-2 from individual cells. J Immunol Methods 125:115-124. 23. Sedgewick JD, Holt PG (1983) Kinetics and distribution of antibody-specific IgE secreting cells during the primary antibody response in the rat. J Exp Med 157:2178-2184. 24. Versteegen JMT, Logtenberg T, Ballieux RE (1988) Enumeration of IFN-y-producing human lymphocytes at the single-cell level. J Immunol Methods 111:25-29. 25. Lewis DB, Prickett KS, Larson A, Grabstein K, Weaver M, Wilson CB (1988) Restricted production of interleukin 4 by activated human T cells. Proc Nat1 Acad Sci USA 85:9743-9747. 26. Multhaupt H, Gross G, Fritz P, Kohler K (1989) Cellular localization of induced human interferon-beta mRNA by nonradioactive in situ hybridisation. Histochemistry 91:315-319. 27. McCall JL, Yun K, Funamoto Y, Parry BR (1989) In vivo immunohistochemical identification of TNFicachetin in human lymphoid tissue. Am J Pathol 135:421-425. 28. Steinmann G, Conlon P, Hefeneider S, Gillis S (1983) Serological visualization of interleukin 2. Science 220:1188-1190. 29. Tabibzadeh S, Poubouridis D, May JT, Sehgal PB (1989) Interleukin 6 immunoreactivity in human tumours. Am J Path01 1351427-433. 30. Barkley D, Feldmann M, Maini RN (1989) The detection by immunofluorescence of distinct cell populations producing interleukin-la and B in activated human peripheral blood. J Immunol Methods 120:277-283. 31. Andersson U, Matsuda T (1989) Human interleukin-6 and tumour necrosis factor alpha production at the single cell level. Eur J Immunol 19:1157-1160. 32. Andersson U, Adolf G, Dohlsten M, Moller G, Sjogren H-O (1989) Characterization of individual TNF-cu and B producing cells after polyclonal T cell activation. J Immunol Methods 123:233240. 33. Palacios R, Martinez-Maza 0, De Ley M (1983) Production of human interferon (Hu IFN-y) studied at the single cell level. Origin, evidence for spontaneous secretion and effect of cyclosporin A. Eur J Immunol13:221-225. 34. Lewis CE, McCarthy SP, Richards PS, Lorenzen J, Horak E, McGee JO’D (1990) Measurement of cytokine release by human cells: a quantitative analysis at the single cell level using the reverse haemolytic plaque assay.J Immunol Methods 127:51-59. 35. Lewis CE, McCarthy SP, Lorenzen J, McGee JO’D (1990) Heterogeneity amongst human mononuclear phagocytes in their secretion of lysozyme, interleukin-1 and type B transforming growth factor: a quantitative analysis at the single cell level. Eur J Immunol 19:2037-2043. 36. Skidmore BJ, Stamnes SA, Townsend K, Glasbrook AL, Sheehan KCF, Schreiber RD, Chiller JM (1989) Enumeration of cytokine secreting cells at the single-cell level. Eur J Immunol 19:1591-1597. 37. Taguchi, T McGhee JR, Coffman RL, Beagley KW, Eldridge JH, Takatsu K, Kiyono H (1990) Detection of individual mouse splenic T cells producing IFN-7 and IL-5 using the enzyme-linked immunospot (ELISPOT) assay.J Immunol Methods 128:65-73.