Simple, sensitive and specific bioassay of interleukin-1

Journal of Immunological Methods, 120 (1989) 271-276 Elsevier

271

JIM05207

Simple, sensitive and specific bioassay of interleukin-1 S t e p h e n J. H o p k i n s a n d M a r i e H u m p h r e y s Unioersity of Manchester Rheumatic Diseases Centre, Clinical Sciences Buildin& Hope Hospital, Eccles Old Roa~ Salford M6 8HD, U.K. (Received 23 January 1989, revised received 3 February 1989, accepted 14 February 1989)

This paper describes a convenient method for the culture of sub-lines of the murine T cell cloned line, D10.G4.1, and the use of these lines in a highly sensitive and specific bioassay for interleukin-1 (IL-1). The cells are cultured with IL-1, interleukin-2 (IL-2), and concanavalin A (ConA), in the absence of feeder cells or antigen. Assays are routinely carried out in the presence of saturating IL-2, which enhances sensitivity and ensures that further IL-2 will not give false positives. Addition of interleukin-4 (IL-4) has a similar effect and can be used together with IL-2 where there is a potential for interference from either cytokine. The assay is not affected by high concentrations of human interleukin-6 or tumour necrosis factor-a (TNF-a) and only minimally affected by high concentrations of murine TNF-et. Key words: Bioassay; Interleukin-1; Interleukin-2; Interleukin-4

IntToduction

Although immunoassays for human IL-1 are becoming increasingly available there is still a requirement for assays which quantitate levels of biologically active material. In addition, immunoassays are not generally available for the determination of IL-1 from other species. The classical assay for IL-1, based on the use of thymocytes, has proved extremely useful in the past but its use has come into question because of its lack of both sensitivity and specificity (Symons et al., 1987; Helle et al., 1988; Ranges et al., 1988; Uyttenhove et al., 1988). Alternative bioassays have been developed and proposed as substitutes, principally because of their increased sensitivity or lack of response to IL-2. We have developed an

Correspondence to: S.J. Hopkins, University of Manchester Rheumatic Diseases Centre, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD, U.K.

improved method for IL-1 assay, based on sublines of the D10.G4.1 (D10) murine T cell line, derived by Kaye et al. (1983). This assay is sensitive to femtomolar concentrations of IL-1 and is insensitive, or can be made insensitive, to cytokines that interfere with other IL-1 bioassays. The characteristics of these cells and the nature of the assay are described here.

Materials and methods

Cytokines and other reagents The source of IL-1 used for the passage of cell lines was dialysed supernatant from activated macrophage-like cell lines. The supernatant source of murine IL-1 used initially was a kind gift of Dr. S. Durum (BRMP, NCI, Frederick, MD) and was derived from lipopolysaccharide (LPS)-activated P388D1 cells as described elsewhere (Tartakovsky et al., 1986). Human IL-1 was derived from the myelomonocytic cell line, THP-1. Preparation of

0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

272 the THP-1 supernatants and purified IL-1 (plL-1) for use as standard laboratory preparations (THP1I and THP-1M), have been described (Hopkins et al., 1988). Generous gifts of cytokines used in these experiments were: recombinant human interleukin-2 (rlL-2) from Sandoz Forschungsinstitut; CM-Sepharose-purified murine interleukin-4 (plL4) derived from the EL-4 thymoma, from Dr. C. Sutton (Celltech); recombinant human interleukin-6 (rlL-6) from Drs. L. Aarden and M. Helle of the CLB in the Netherlands; recombinant human tumour necrosis factor-a ( r H u T N F - a ) from BASF/Knoll, F.R.G.; and recombinant murine T N F - a ( r M u T N F a ) from the National Institute Biological Standards Control (NIBSC), South Mimms, U.K. The cytokine units used were referenced to the NIBSC interim standards for I L - l a and IL-lfl (86/632 and 86/552, 10 pg = 1 U), the BRMP interim standard for human IL-2 and recombinant murine IL-4 from Genzyme (108 U / m g ) . Purified IL-4 was used in preference to recombinant IL-4 for most experiments since it was available at the greater concentrations required to demonstrate response saturation. RPMI 1640 media (Northumbria Biologicals), supplemented with 2 m M glutamine, 10% foetal calf serum (Sera Lab), 5 × 10-5 M 2-mercaptoethanol (BDH) and gentamicin sulphate at 50 U / m l (Nicholas), was used routinely. Concanavalln A (ConA) was grade IV from Sigma.

Cell lines The D10 cells were originally obtained from Dr. S. Durum, who received them as a gift from Dr. J. Kaye (Yale, CT). The cells had initially been passaged on feeder layers of syngeneic mitomycin C-treated spleen cells with 100/~g/ml conalbumin (Kaye et al., 1983). The cells were transferred into media supplemented with 5% P388D1 supernatant and 5 # g / m l ConA, but no feeder cells or conalbumin. These cells grew extremely slowly and it was necessary to maintain them at a high density. At 40 days the cells were frozen in 30% FCS, 10% dimethylsulphoxide. Experiments with cells that were not frozen at that time, indicated that the rate of proliferation could be greatly increased by addition of IL-2. The frozen cells were subsequently thawed into media

similar to that described above, but supplemented with rIL-2. The IL-2 was maintained at approximately 10-40 U / m l at time of passage. The source of supernatant IL-1 was changed to the THP-I-derived material and was used at approximately 2 - 4 U / m l . The ConA concentration was also standardized at 3 /~g/ml. The cells were routinely subcultured, twice weekly, by diluting with fresh media (usually 1/10). A number of sub-lines were generated in this way and some have manifested rather different growth factor requirements. The line primarily described here was designated D10(N4)M (D10N) and has been in continuous culture for more than 2 years.

Proliferation assays Thymocytes were prepared from the thymuses of C 3 H / H e J mice from our own animal unit, using scalpel blades to gently break apart the tissue. Cytokine preparations were serially diluted in 96-well microplates. Cells were then added to give 1 × 10 4 D10N or 8 × 105 thymocytes per culture (200/xl volume). The cells were incubated for 1-3 days and labelled by the addition of 37 kBq [3H]thymidine (TRA.61, 185 G B q / m m o l , Amersham) for the last 4 or 18 h, before harvesting onto glass fibre filters with distilled water (Skatron harvester) and counting in a conventional liquid scintillation cocktail.

Results

Synergistic enhancement of the IL-1 response by IL-2 Activation of D10N by IL-1 was weak or absent unless ConA was present (Fig. 1). There was some response to IL-2 alone but as the concentration of IL-2 was increased the response to IL-1 was also increased until the concentration of IL-2 becomes optimal. The addition of optimal or supra-optimal IL-2 therefore permits assays which are both increased in sensitivity to IL-1 and insensitive to further IL-2. ConA was not absolutely required in this situation, but the response was usually increased in its presence. We therefore routinely performed the assays with optimal ConA ( 3 - 5 / t g / m l ) present.

273 although the concentration of IL-1 generating a 50% response varied slightly from one assay to another, the relative values between IL-1 preparations and the standards remained stable.

a) 100--

Synergy with IL-4 As with many other IL-l-sensitive murine T cell lines the D10 cells were also responsive to murine IL-4, particularly in the presence of IL-1 (Kupper et al., 1987; Lichtman et al., 1987a). Although this was not a problem when evaluating samples derived from human sources it is a potential limitation when evaluating samples from other species. We therefore determined whether we could saturate IL-4 responsiveness as had been done for IL-2. This was indeed possible, as shown in Fig. 3a. Furthermore, a saturating concentration of IL-2 and IL-4 could be used together (Fig. 3b), both to increase sensitivity and eliminate potential interference from either cytokine.

75-cpm x 10-3

50--

25

o

b)

10ox- ~ ~

e4~mx

O-

~ 75-

a50--

o

o'.5 1.4

i

17

li0

I L - I (u/ml)

Fig. 1. Synergy of IL-1 and IL-2. Recombinant I L - l a was titratexl in the presence of D10N cells, without (a), or with (b) 3 # g / m l ConA. IL-2 was either absent (o), or present at 1

25--

O/ml (e), 3 O/ml (A), 10 U/ml (A), 30 U/ml (r7) or 100 U/m] (B).

x~ O--

Sensitivity of the assay The response of thymocytes and two D10 sublines is compared in Fig. 2. Both D10 sub-lines shown were significantly more sensitive to IL-1 than the thymocytes. Although one sub-line was more sensitive than the other, both lines assigned similar values to unknown samples when compared to a standard IL-1 preparation. Similarly,

Y

I

I

I

I

I

T

I

I

I

6600

2200

750

250

50

30

10

3

1

Units I L - I x 10.3

Fig. 2. Comparison of the thymocyte and D10 N methods of IL-1 assay. Recombinant I L - l a was titrated in a thymocyte ( × ) , D10(N4)M (o), or D10(N4)S ( o ) cell assay for IL-1 and the results plotted with respect to the maximum response in each assay. The background minimum and maximum responses in each assay were respectively, 1408 and 53074 clam (thymocytes), 2413 and 164835 cpm (D10(N4)M), and 46910 and 287350 cpm (D10(N4)S).

274

b)

250--

a) 175200-150-

cpm x 10 -3 125-

150--

cpm x 10 -3 100-

100-75-

5050-25--

0~ ,I 0

I 0.02

I 0.07

1 0.2

I 0.6

I 1.9

1 5.6

0

I 0.04

I 0.14

I 0.4

I 1.2

I 3.7

I 11

I 33

I L - I (u/ml) I L - I (u/ml)

Fig. 3. Synergy of IL-1 with IL-2 and IL-4. Serial dilutions of pIL-1 were prepared in the presence of (a) either saturating (30 U / m l )

IL-2 (A), or plL-4 at 0 U/ml (x), 23 U/ml (o), 70 U/ml (O) 209 U/ml (D), 629 U/ml (11)or 1889 U/ml (zx); (b) either saturating IL-2 alone (-) or saturating IL-2 together with plL-4 at 209 U/ml ( × ), 629 U/ml (a) or 1889 U/ml (o).

Effect of other cytokines Although the D10(N4)M cells were extremely sensitive to IL-1, recombinant h u m a n IL-6 and recombinant human T N F - a did not stimulate [3H]thymidine incorporation (Fig. 4). Neither did we find any evidence of synergy between these cytokines and IL-1 (not shown). Murine T N F - a induced a small but reproducible increase in [3H]thymidine incorporation (between 5 n g / m l and 15 n g / m l ) .

Discussion

The thymocyte assay has been a valuable tool for evaluating IL-1 activities but its relative insensitivity to IL-1 and its sensitivity to other cytokines severely limit its usefulness ( Symons et al., 1987; Lotz et al., 1988; Ranges et al., 1988). In particular its sensitivity to IL-6 may have led to

the mis-identification of IL-l-like activities in situations where it subsequently became apparent that IL-6 was the predominant cytokine (Helle et al., 1988). The use of D10 cells for the assay of IL-1 was described by Kaye and Janeway (1984) as a more sensitive alternative to the thymocyte assay with the advantage over other contemporary assays (Gillis and Mizel, 1981; Conlon, 1983) that it involved the use of only one cell line in a single culture step. Subsequently D10 cells formed the basis of one of the most popular alternative IL-1 assays, but have probably not been more widely used because of the difficulty of maintaining stable cell lines on syngeneic antigen presenting cells. In addition, although the cells have not been reported to respond to m a n y cytokines, they do, in c o m m o n with a number of other IL-l-sensitive T cells, respond to IL-2 and murine IL-4. We have overcome the difficulties of maintaining the cell line by culturing them continuously in conditions

275

200 -

/

Gpm x 10 -a

150-

100 -

/

50-

0

J/ //

/

/

I

I

I

I

I

I

I

10 -z

1

101

10 2

10 3

10 4

lOS

Cytokine concentration (pg/ml)

Fig. 4. The effect of T N F and IL-6 on D10N cells. D10N cells were cultured, either alone (11), or together with the indicated concentrations of r I L - l a ( × ) , r H u T N F - a ( ~ ) , r M u T N F - a (O) or rIL-6 (o), in a standard assay with 30 U / m l IL-2 and 3 /~g / m l ConA.

which are similar to those of the assay. Similar maintenance of D10 cells, in the absence of accessory cells and antigen, has recently been reported by Campos-Neto and Stashenko (1988). It should be noted that some of our D10 sub-lines are capable of responding to IL-1, IL-2 or ConA alone. The D10(N4)S sub-line now responds in this way (unpublished observations). Such transformations of D10 cells have been described elsewhere, to occur either spontaneously (Lacey et al., 1987) or as a consequence of viral infection (Lichtman et al., 1987b). The D10(N4)M sub-line has, however, remained relatively stable, both in our hands and, to our knowledge, in other laboratories to which it has been distributed. Each of our D10 sub-lines, cultured in this way, retains H-2restricted responsiveness to conalbumin (not shown). Having selected a poor IL-2 responder we exploited this attribute, and the synergy between IL-1 and IL-2, both to increase the sensitivity of the assay and to saturate the IL-2 response. When

it became apparent that murine IL-4 also activated T cells we found that we could use a similar protocol of saturation to overcome the problem. Although the 'background' [3H]thymidine incorporation is increased when saturating IL-2 a n d / o r IL-4 is added this makes no difference to the quantitation of IL-1, even when the background is increased considerably. Saturation of the thymocyte assay with IL-2 has recently been reported as a method to avoid IL-2 interference in this assay (Falk et al., 1987), although the sensitivity to IL-1 was not increased. A similar approach has been employed in another situation where a cell line showed dual responsiveness to IL-3 and IL-4 (Mosmann et al., 1986). The other potentially interfering cytokines are T N F (Ranges et al., 1988) and IL-6 (Helle et al., 1988; Uyttenhove et al., 1988). These cytokines either do not interfere, or only minimally affect the assay, at any concentrations we have tested, or which are likely to be encountered in physiological fluids or cell supernatants. Two other sensitive IL-1 assays have recently been described, which are refractory to IL-2. One of these involves the use of two cell lines and two stages of culture, which introduces a further level of difficulty (Gearing et al., 1987). This line has also recently been found to respond significantly to murine and human T N F - a at concentrations of 1 n g / m l and 10 n g / m l respectively (A. Gearing et al., personal communication). The second method, based on IL-2 receptor induction by IL-1, appears to respond to IL-4 and T N F to a hmited extent and does involve some additional technical complexity and time at the stage of IL-2 receptor quantitation. Having developed and used the assay described here over a number of years and evaluated a variety of problems encountered in other systems, we believe that its relative simplicity, sensitivity, specificity and reliability make it an extremely valuable tool for determining levels of bioactive IL-1.

Acknowledgements

We are most grateful to the persons indicated in this paper for their very generous gifts of cyto-

276

kines and to the North Western Regional Health Authority for financial support.

References Campos-Neto, A. and Stashenko, P.P. (1988) Interleukin 1 and T cell activation. Immunol. Today 9, 131. Cordon, P.J. (1983) A rapid biological assay for the detection of interleukin 1. J. Immunol. 131, 1280. Falk, W., Kammer, P.H. and Mannel, D.N. (1987) A new assay for interleukin-1 in the presence of interleukin-2. J. Immunol. Methods 99, 47. Gearing, A.J.H., Bird, C.R., Bristow, A., Poole, S. and Thorpe, R. (1987) A simple sensitive bioassay for interleukin-1 which is unresponsive to 10 a U / m l of interleukin-2. J. Immunol. Methods 99, 7. Gillis, S. and Mizel, S. (1981) T cell lymphoma model for the analysis of interleuldn-l-mediated T-cell activation. Proc. Natl. Acad. Sci. U.S.A. 78, 1133. Helle, M., Brakenhoff, J.P.J., DeGroot, E.R. and Aarden, L.A. (1988) Interleukin 6 is involved in interleukin 1-induced activities. Eur. J. Immunol. 18, 957. Hopkins, S.J., Humphreys, M. and Jayson, M.I.V. (1988) Cytokines in synovial fluid. I. The presence of biologically active and immunoreactive IL-1. Cfin. Exp. Med. 72, 422. Kaye, J. and Janeway, Jr., C.A. (1984) Induction of receptors for interleukin 2 requires T cell Ag: Ia receptor crosslinking and interleukin 1. Lymphokine Res. 3, 175. Kaye, J., Porcelli, S., Tite, J., Jones, B. and Janeway, Jr., C.A. (1983) Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen presenting cells. J. Exp. Med. 158, 836. Kupper, T., Horowitz, M., Lee, F., Robb, R. and Flood. P.M. (1987) Autocrine growth of T cells independent of inter° leukin 2: identification of interteukin 4 (IL-4, BSF-1) as an

autocrine growth factor for a cloned antigen-specific helper T cell. J. Immunol. 138, 4280. Lacey, D.L., Chappel, J.C. and Teitelbaum, S.L. (1987) Interleukin 1 stimulates proliferation of a nontransformed T lymphocyte line in the absence of a co-mitogen. J. Immunol. 139, 2649. Lichtman, A.H., Kurt-Jones, E.A. and Abbas, A.K. (1987a) B cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 84, 824. Lichtman, A.H., Williams, M.E., O'Hara, J., Paul, W.E., Failer, D.V. and Abbas, A.K. (1987b) Retrovirus infection alters growth factor responses of T lymphocytes. J. Immunol. 138, 3276. Lotz, M., Jirik, F., Kabouridis, P., Tsoukas, P.C., Hirano, T., Kishimoto, T. and Carson, D.A. (1988) B cell stimulatory factor 2/interleukin 6 is a costimulant for human thymocytes and T lymphocytes. J. Exp. Meal. 167, 1253. Mosmann, T.R., Bond, M.W., Coffman, R.L., O'Hara, J. and Paul, W.E. (1986) T cell and mast cell fines respond to B cell stimulatory factor 1. Proc. Natl. Acad. Sci. U.S.A. 83, 5654. Ranges, G.E., Zlomik, A., Espevik, T., Dinarello, C.A., Cerami, A. and Palladino, Jr., M.A. (1988) Tumor necrosis factora/cachectin is a growth factor for thymocytes. Synergistic interactions with other cytokines. J. Exp. Med. 167, 1472. Symons, J.A., Dickens, E.M., DiGiovine, F. and Duff, G. (1987) Measurement of interleukin-1 activity. In: M.J. Clemmens, A.G. Morris and A.J.H. Gearing (Eds.), Lymphokines and Interferons. IRL Press, Oxford, p. 269. Tartakovsky, B., Kovacs, E.J., Takaks, L. and Durum, S.K. (1986) T cell clone producing an IL-l-like activity after stimulation by antigen presenting cells. J. Immunol. 137, 160. Uyttenhove, C., Coulie, P.G. and Van Snick, J. (1988) T cell growth and differentiation induced by interleukin-HP1/IL6, the murine hybridoma/plasmacytoma growth factor. J. Exp. Med. 167, 1417.