An antiproliferative bioassay for interleukin-4

Inrerleukin-4 (IL41 is cwremly being used for therapeutic inlervention in a wide range of malignan direares as an antitumwr ageor Altbagb bioassayshave been developedmar measuretie pmlifemrivecapaciryof IL-4. none measurethe antiproliferativetiviry of this mokcuk. We have developed a simple. sensitive bioassayfor human IL-4 based on the ability of this ~ytokincto inhibit the proliferationof the humzmlune,carcinomaline, CCL-l85 M easy to maintain.cymkine i&&&m. cell line. It is rapid. ~repmducibleand sensitive. able to detect 2 pg/ml IL-4 The assay is completely muqwnsive to atl aher imertcuki~ from n-2 to IL-12, to ti colony stimulating factors and hansforming growth factor baa and is 10%fold less sensitive to imaferon-o olmour wrosir factor-a. IL-Ip and IL-13. The assay can be made completelyspexitii for n-4 by incmdingspecific neutralizingantibodiesfor IL-4 a-4 in suitable fo: tire estimationof IL-4 m boyl plasma ard serum sampler

1. Inlmduction

Interleukin-4 (fL-4) is an I S-20 kDa glycoprotein mainly prnauced by subs& of the T lymphocyte

lineage arId mast cells (Howard et al., 1982; Callard. 1991). It has ao extremely diverse range of biological activities on a wide range of cells (Spits, 1992; Ohara et al.. 1987) acting as a costimulator of B cell proliferation and differentiation (Paul, 1991), stimulates T cell proliferation (Yokota et al.. 1986). affects haematopoiesis (Brameyer et al., 1988; Peschel et al., 1987). monocyte function (Hart et al., 1989) and the development of lymphocyte activated killer cells (Phillips et al., 1992). Although IL-4 can stimulate the proliferation of a wide range of cell types (Oham, 1988). it also has the ability to inhibit the growth of several different types of turnour cells such a6 lung twoo~rs, renal cell carcinomas and B lymphomas (Golumbek et al., 1991; LIefrance et al.. 1992; Toi et al., 1992; Taylor et al.. 1990). This has

led tic. Yu al..

to the widespread inve+gation of the tberapeuanticancer potential ot IL-4 (Tsu et al.. 19% et al.. 1993; Mqolin et al.. 1994; Comacoff et 1992; Pwi id Siegel, 1993; Topp et al., 199%). Although several b&assays exist for maming

IL-4, “hey measure

either the proliferative

CO? 3T-C. Cultores were passaged when they reached a density of 2 X IO5 cells/ml and diluted to a density of 5 X 10’ cells/ml. These ceBs have bea shove

to possess

approximately

capacity

of IL-4 or its ability to upregulate surface markers. IL-4 can be measured by tbe ability to stimulate the _mulh of tbe M07e megakaryoblastic cell line (Komatsu et al.. 1991). rhe TFI erytbmieukaemic

rhiL-f

ceil line (Kitamura

et al.. 19s9) or the human

were gifts from R&D

receptw

murine cell line, CTh4S

traosfected

UXM high aftinity

binding sites for IL-4 (Topp et al., 199%)

IL-4

iHu-Li

Cymkiner

uxxe gewaously

to mn-10

from Genetics

dooated

and nerve

gmwth

Systems,

instiNt%

as follows: factor (NGF)

ML-1 1 and rhIL-12

IL-13 horn Sattofi, rhGM-

et al.. 19893. None of these cell liis are specific for IL4 as all proliferate to a variety of other cyTokines and all are affected by inhibitory cytokioes (Wadhwa et al.. 1991). IL4 can also be measured by its ability

CSF fmm Scbsring Plwgh. rhGC.SF and rb-ery;xropoietin (rhEpo) and stem cell facts tSCF) from Amgen. rM+CSF from Cbiroo. rb -mmour necrosis factor a GhTNF-a) from Gemmech, rh-interfenm-a

to upregulate

CD23

on Ramos

cell

(rbiM-a)

lines

flow

cytofluorimetry

and

tom 6

using

B lymphocyte (Siegel

Mostowki, 1990) or flu-nre immunoassay (Custer and Laze, 1990). but these assays are either not very sensitive or not suitable for the rapid tbmughput of larger numbers of samples (e.g. dilw tioo series). It is not known how fL-4 is able to induce either proliferation

or inhibition

depending

upon

sensitive

to dze amipmliferative

Rocbe, rb-uansfonning

rhTGF-& M) from nmopbic leukaemia

tim

Boebringer

gmti

fat-

Ingellteim,

from CeiloUr. tb-tatin M (rhGnco Bristol Myers Squibb, rh-ciiiq neufactor (IbCNTF) horn syxrgen, minhibitoq factor (rbLIF) fmm Smdoz.

rbe cell

type on which it acu nor wbetbu measurements of the proliferative capacity of the cytokine relates to its

is paniculariy

fmm

t&TGF-&)

A MAb to IL4 was produced in BALB/c mice as described previously (Bird et al.. 1991). The MAb recognizes all recombinane forms of IL4 and is oeuualizing in bioasaya.

effects

of iL-4 {Topp et al., 1993) and we have developed an antiproiiferative assay for IL4 that is rapid, easy to perform. appqxiate fw many sample types (plasma, senmi or ruitwe supzm&xts) and ioseostive to all otbxr proliferative cyxokines vstcd. er.cept IL-13

2. I. Ceils and maimemamce The

human

long

carcinoma

ceil

line

CCL185

(Giard et al.. 1983) was obtained from the American Type Cultwe ColIection. The cells wx grown in HAM’s Fl2K medium (Gibco) supplemented with 10% fortal calf serum (FCS) and penicillin/strepccmycin in a homiditied atmosphere containing 5%

Dilutions for the consmlcti3n of IL-4 dose Itspmlse carves and a.ssa,x of srmp:es weoz prepared by addition of 40 ~1 of sampbze/waw&td to 160 pl of HAM’s F12K ox&am containing 10% FCS and antibiotics into tlx 6% two aizlls of altemafe rows of a microzioe plate. Ail additional wells of the plate coma&d 100 ~1 of HAM’s/FCS. A two-fold diiutiooseriesws~ by ramving Irl ~1 from the first two %vells ptepared as &&bed abow and clansfening the material to the lbii and four& wei2c cf the same row. The sample/stza&rd was mixed

of the microtitre piate with tbe last 100 ~1 being discankd to pro&a the sample/stmdard dilution series in 100 ~1 aliquots/well. Additional negative

controls containing 100 &I of medium alone were included. CCL185 cells were used when tky reached a cell density of 2X 10’ cells/ml. The cells were washed twice in HAM’s medium and resuspended in HAM’s contaming 10% FCS, 100 pg/ml streptomycin and 100 U/ml penicillin at a density of I x IO’ cells/ml. 100 *I aliqilots of tbc cell suspension were then ad&d to the previously prepared micmtim plate cotttalning the dose response curve and sample dilutvm series. Tk microtitre plate was incubated at 37T in a 5% CO, humidified atmosphere for 16 h. To assess cell pmlifcmtion, OS &i of [‘Hkhymidine

([‘HJTdr)

was added/well

and the

microtitre plate returned to the incubator for a further 4 II. The cells arc then harvested onto filta mats and the radioactivity incorporated into DNA was measured using a scintillation counter. Statistically valid quantification of the levels of IL-4 in the samples was made ty comparison of the sample dilution series to tbe dose response curve of the WHO International standard for IL-4 (88/656) using parallel line analysis (Eurapcan Pharmacopoeia, 1971). The sensitivity of this and other assays discussed in this manuscript was defined as the lowest level un the linear portion of the cwve that was statistically valid ( p < 0.05).

133

for IL-4 is raptd. sensirive,easy IO perform with large numbers of samplesand can bc made specific for IL-4 with the use of neutralizing antibodies10 IL-4.

Acknowledgements We thank Mrs. Debarah Richards for processing the manuscript.

Gixd. D .I. AamNO”. S.A.. Tcdam. cc.,..Amnem. P.. ixerrey. J.H.. Dasik. H. and Park. W.P. (1983, I” YiUO C”klYalicm or

mumxhmpy 12. 82. Wadbwa. M.. Dilw. P.. Tubbr. 1.. Barmwcbffe. T.. Mabon, B.