Download Quantitative Receptor Binding Assay of Interleukin

Mol. Cells, Vol. 2, pp. 335-340
Quantitative Receptor Binding Assay of Interleukin-l Using a Flow
Cytometer
Yun Soo Bae, Eun Hee Lee l , Kilhyoun Kim I, In-Seong Choe l * and Tai-Wha Chung
Laboratory of Immnochemistry and 1Laboratory of Cell Biology, Genetic Engineering Research
Institute, Korea Institute of Science and Technology, Taejon 305-606, Korea
(Received on August 25, 1992)
Interleukin-l (IL-l) is a cytokine that mediates various immune responses and inflammation. IL-l binds to its receptor molecules expressed on the responding cells to mediate
these responses. We prepared fluorescein-labeled IL-l , and established a quantitative assay
for receptor binding using a flow cytometer. Mouse thymocytes expressing IL-l receptor
molecules on their surface were employed in the assay. With this binding assay, we obtained IL-l binding proportional to the quantity of IL-l in the range of 2.5 to 100 units
at a constant thymocyte number. This assay worked at the temperature between 25 and
45 °C. This assay appeared to be more rapid, convenient and simple than the conventional
binding assay using 125I-labeled IL-l and thymocyte proliferation assay using phytohaemagglutinin as a coactivator. This method could be utilized in other researches regarding
IL-l activity or IL-l receptor expression. Thus, IL-l inhibitors isolated from febrile patient
urine were analyzed by this method. IL-l receptor expression on mouse thymocytes along
with expressions of other surface markers was also studied.
Interleukin-l (IL-l) has a broad spectrum of biological activities; IL-l induces the proliferation and differentiation of a diverse array of cell types including
lymphocytes, macrophages, endothelial cells, fibroblasts and synovial cells, and stimulates them to secret
numerous physiologically-active agents which might
elicit further cellular responses (Oppenheim et al.,
1986; di Giovine and Duff, 1990). Its target cells also
include Band T cells. It has been reponed that there
are two different IL-l proteins with diffe]ent isoelectric
points (PI) (Gubler et al., 1986; Auron et al., 1984; March et al., 1985; Lomedico et al., 1984). The acidic form
(pI 5.0) has been designated as IL-Ia and the neutral
form (PI 7.0) IL-I~. Although they have limited sequence homology, two types of IL-l molecules seem to
act very similarly (Wood et al., 1985).
IL-l exerts its cellular functions by binding to its
specific receptors expressed on target cells, and the
nature of the receptor molecules has recently been
reported (Dower et al., 1985; Sims et al., 1988; MacDonald et al., 1986; Dinarello et al., 1989). Since binding
of IL-I molecules to their receptors would reflect their
physiological activity, the binding of IL-l might be
measured in IL-l assay instead of measuring its functional activity as in its conventional bioassay that
measures proliferation of murine thymocytes (Liao et
al., 1984). The thymocyte proliferation assay was established based on the fact that the thymocytes respond
to IL-l to proliferate in dose-dependent manner in
the presence of lectins such as phytohaemagglutinin
(PHA).
* To whom correspondence shoud be addressed
Receptor binding assay of IL-I employing 125I-labeled IL-I has been reported (Dower et al., 1985). It
would be advantageous if fluorochrome-labeled IL-I
can be used in replace of radioisotope-labeled IL-I
in receptor binding assay to monitor the binding of
IL-l, since it produces no radioactive wastes, and
moreover, when using a flow cytometer for fluorescence measuring, one might enjoy a computer-supported
diversity in analyzing the data. Detection of receptors
for IL-l as well as IL-2 and IL-4 by using fluorochrome-labeled ligands have been previously described
(Tanaka et al., 1989; Harel-Bellen et al., 1989; Zuber
et al., 1990). However, these reports did not constitute
quantitative determinations of ligands, interleukins.
In this article, we report a quantitative receptor binding assay for IL-l established by using fluorochromelabeled IL-I and a flow cytometer for measuring the
fluorescence intensity. We propose that this assay
could substitute for the receptor binding assay employing 125I-labeled IL-l as well as thymocyte proliferation
assay. This assay was utilized to measure IL-I receptor
expression and IL-I activity which would be reflected
by fluorescence intensity bound to the target cells. It
was also applied to measure activity of IL-l inhibitors
in the samples isolated from human febrile urine,
which appeared to block IL-l action in a competitive
manner (Seckinger et al., 1987).
The abbreviations used are: FITC, fluorescein isothiocyanate;
IL-I , interieukin-I; MFI, mean fluorescence intensity; PHA
phytohaemagglutinin.
© 1992 The Korean Society of Molecular Biology
336
Interleukin-l Assay by Flow Cytometl)'
Materials and Methods
Mouse thymocyte proliferation assay of IL-J
Thymi were removed from C 3H/HeJ mice, and single cell suspension of thymocytes were prepared. The
cells were washed with RPMI 1640 (Sigma Chern. Co.,
St. Louis, MO) and resuspended in the same medium
supplemented with 10% heat-inactivated fetal calf serum (FCS) , 300 ~glml glutamine, 5 X 10- 5 M 2-mercaptoethanol, and antibiotics. Thymocytes were cultured for 72 h in the presence of 1 ~g1ml phytohaemagglutinin (PHA; Difco Lab., Detroit, MI) and human
recombinant interleukin-l ~ (IL-l~; Genzyme, Boston,
MA) according to the method described by Liao et
al. (1984). The cultures, pulsed with 0.5 ~Ci [ 3HJthymidine (Amersham, Buckinghamshire, UK) after 48
h, were halVested and counted on a liquid sciI\l.illation
counter.
Mol. Cells
1990), and the nature of the receptor molecule has
well been .described (Dower et al., 1985; MacDonald
et al., 1986; Sims et ai., 1988; Dinarello et ai., 1989).
In this study, prepared were fluorescein-conjugated
human recombinant IL-l~ by mixing the IL-l~ with
fluorescein isothiocyanate (FITC), and used to monitor
the binding of IL-l molecules to their receptors expressed on target cells, thymocytes. The FITC-coupled
IL-l~ (IL-l-FITC) prepared in this way retained the
biological activity when tested by thymocyte proliferation assay (data not shown).
For receptor binding assay, aliquots of IL-l-FITC
were added to a suspension of thymocytes and incubated, before the thymocytes were analyzed on a flow
cytometer. Figure lA shows increase of fluorescence
intensity with increasing amount of IL-l-FITC, which
300~--------------------------,
A
Preparation of fluorescein-labeled JL-J S (IL-J-FITC)
IL-l ~ was dissolved in 50 mM sodium carbonate
buffer (pH 8.5) containing 75 mM NaC!. Added was
1.4 mg Celite-10% fluorescein isothiocyanate (FITC;
Sigma Chern. Co., St. Louis) to each mg of IL-l~,
and incubated at 4 °c for 3 h. Unreacted Celite-10%
FITC was removed by centrifugation, and the supernatant collected was dialyzed and stored at - 20 °C
until used.
Receptor binding assay of /L-J
Thymocytes prepared from C 3H/HeJ mice were washed twice with Hank's balanced salt solution (HBSS)
supplemented with 2.5% FCS. Each assay mixture contained 1 X 106 thymocytes and IL-l-FITC of indicated concentrations in 0.2 ml HBSS. After each assay
mixture was incubated for 1 h, unbound IL-l-FITC
was removed by centrifugation. The cells bound by
IL-l-FITC were analyzed on a flbw cytometer (BectonDickinson, San Jose, CA) to measure the fluorescence
intensity.
Partial purification of IL-J inhibitor from human f ebrile
urine
Urine collected from febrile patients was clarified
by filtration on filter papers and concentrated by a
protein concentrator (Pellicon, Millipore Co., Bedford,
MA). The concentrated urine which had been dialyzed
against phosphate-buffered saline (PBS, pH 7.4) was
fractionated by ammonium sulfate. The precipitate
from the solutiori 40-80% saturated with ammonium
sulfate was dissolved in 1/10 of the original volume
of PBS. The resulting solution was dialyzed against
PBS and stored at - 20 "c until used.
Fluorescence intensity
1 . 5~---------------------------'
B
1.0
0 . 5i-~--~-r--r-~-.r-~~--~~
0.0
0 .5
1.0
1.5
2 .0
2.5
Log [IL-I-FITC]
Results
Figure 1. Flow cytometric analysis of IL-l-FITC binding to
It is well established that IL-l exerts its biological
functions by binding to specific IL-l receptor molecules expressed on the responding cells (Gubler et al.
1986: Oppenheim et al., 1986; di Giovine and Duff,
murine thymocytes. (A) Histograms of fluorescence intensities obtained by adding 0 (-), 25 (oo.), 50 (---) and 100 (---)
units of IL-l to thymocytes. (B) Plotting of mean fluorescence intensities against IL-l quantities added in logarithmic
scales.
Yun Soo Bae et al.
VoL 2 (1992)
reflects increasing quantity of IL-1-FITC bound to the
cells. When logarithms of mean fluorescence intensity
(MFI) values were plotted against logarithms of IL-1FITC quantities used, a linear relationship between
the two parameters was obtained in the range of 2.5
to 100 units of IL-1 with a given number of thymocytes (Fig. lB).
It was also possible to measure the binding of unlabeled IL-1 molecules in the assay mixture by using
IL-1-FITC of a known quantity, the binding of which
would compete with unlabeled IL-I molecules. It
should result in the decrease of MFI, and from that
the quantity of unlabeled IL-I in testing samples could
be calculated. As shown in Figure 2, MFI which represents the binding of IL-l-FITC decreased with increasing amount of unlabeled IL-1 that competed for
the receptor molecules, and the decrease of MFI reached a plateau with an excess amount of unlabeled
IL-1 .
Since the fluorescence intensity, i.e., the quantity of
IL-1 bound to its receptors, should greatly be affected
by the number of receptor molecules, it would be necessary to examine the effect of changes in thymocyte
numbers on the outcome of the assay. In thymocyte
proliferation assay, the magnitude of the response increased as the number of thymocytes used in the assay
was raised; the maximal response was obtained with
about 5 X 105 cells per well, and the response decreased with a higher number of cells in the culture (Fig.
3). In receptor binding assay, on the other hand, MFI
decreased with increasing number of cells. Assuming
that each thymocyte contains a constant number of
receptor molecules, raising the cell number should
337
cause decrease in the average number of IL-1 molecules bound to each thymocyte at a given IL-I quantity. The logarithms of the two parameters showed a
linear relationship in the range of I - 10 X 106 cells
per tube, as shown in Figure 4.
Although the 37 °C temperature is strictly required
for the thymocyte proliferation assay since the cells
must be cultured at least for 3 days, in receptor bind-
30,------------------------------,
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20
0..
u
X
"S
10
o
40
20
60
100
80
120
10- ' X Number of thymocyte
Figure 3. Effect of cell number on the thymocyte proliferation assay. Thymocytes of indicated number were cultured
with 25 units of IL-I and I /lg/ml of PHA before pulsed
with 0.5 /lCi [lH]thymidine, and counted.
30,--------------------------------,
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o
20
Unlabeled murine
40
60
IL-I~
Figure 2. A competitive binding of IL-I-FITC in the pres-
ence of unlabeled murine IL-l. Twenty-five units of IL-IFITC were mixed with 1 X 106 thymocytes in the presence
of unlabeled IL-I (1 , 2.5, 10, 25 and 50 units), and the thymocytes were analyzed on a flow cytometer (plotting in logarithmic scale is shown in the inset).
0 .50 ~--~--~--~--r_--r-_,r_~--_;
5.5
6 .0
6.5
7.0
7.5
Log [cell number]
Figure 4. Effect of cell number on IL-I-FITC binding to
thymocytes analyzed by a flow cytometer. Two units of ILI-FITC were mixed with I X 106, 2 X 106, 5 X 106, or
I X 107 thymocytes, and MFI were plotted against the number of cells in logarithmic scales.
338
Interleukin-l Assay by Flow Cytometry
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lcr
1(f 0l------------------------~
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Figure 5. Effect of temperature on IL-I-FITC binding to thymocytes. Twenty-five units of IL-l-FITC were mixed with
1 X 106 thymocytes at the temperature indicated in the absence (_) or in the presence (0 ) of urine-derived inhibitor.
goo
lQ3
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...., 102
30
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Figure 7. Dual analysis of thymocytes; expression of IL-l
receptor along with other surface markers. Thymocytes were
·stained with IL-J-FITC and monoclonal antibody Jlj.1O conjugated with duochrome (A). or with IL-l -FITC and monoclonal antibody Jlld.2 conj ugated with duochrome (B).
20
Urine (J.1l)
Figure 6. Inhibition of IL-l-FITC binding to thymocytes by
urine-derived IL-l inhibitor. Twenty-five units of IL-I -FITC
were mixed with I X 106 thymocytes in the presence of
the inhibitor of increasing quantity. MFI was plotted against
IL-l quantity used in logarithmic scales.
ing assay which takes less than 1 h for binding, the
temperature can be varied. In fact, it appeared that
the amount of bound IL-I -FITC increased as the reaction temperature was raised (Fig. 5). The maximal
binding of IL-l -FITC was obtained at 45 °c, and this
temperature did not give any disadvantage to this assay as long as the IL-l binding was concerned. Limited binding of IL-l -FITC to thymocytes was observed
at 4 "c , probably due to low association constant (Dower et at., 1985). Note that a sample containing IL- \
inhibitor prepared from human febrile urine (see
below) substantially lowered the IL-l binding, to about
the same level at all the temperatures tested (Fig. 5).
The receptor binding assay of IL-\ was applied to
another study regarding assay of IL- l inhibitors. A
fraction containing IL-l inhibitor activity was isolated
from human febrile urine, and analyzed by the receptor binding assay. When IL-l-FITC was added to thymocytes in the presence of the IL-l inhibitor, the IL-l
binding to the cells was inhibited in dose-dependent
manner, and the inhibition reached a plateau with
an excess amount of the inhibitor (Fig. 6). These results suggested that the inhibitor blocked the IL-l
binding to the cells specifically and in a competitive
manner.
Vol. 2 (1992)
Yun Soo Bae et al.
The receptor binding assay also provided a way to
analyze the IL-l receptor expression on the cell surface along with other markers on the same cells. Thymocytes prepared from C 3H/HeJ mice were stained by
IL-l-FITC along with a monoclonal antibody Jlld.2
(Bruce et at., 1981) which is reactive with an epitope
expressed on murine B cells and immature thymocytes, or another monoclonal antibody Jlj.lO (Bruce et
at., 1981) which is reactive with thy-l antigen on thymocytes and T cells. Most cells turned out to be positive for both IL-l and Jlj.lO which comprised 95%
of total cells, demonstrating that thymocytes expressing
high level of thy-l molecules expressed high level of
IL-l receptors (Fig. 7A). However, expression of Jll d.2
marker molecules among thymocytes expressing IL-l
receptor divided the population into two groups; a
group strongly positive for Jll d.2 comprised 65% of
total cells, and the other group weakly positive or negative for Jlld.2 comprised 30% of the total (Fig. 7B).
Discussion
There are a few different ways to measure IL-l activities. The most commonly used would utilize the fact
that IL-l stimulate thymocytes to proliferate in dosedependent manner. Addition of lectins like PHA
usually increases the sensitivity of the thymocyte response elicited by IL-l. However, thymocyte proliferation which is ~sually measured by determining [ 3HJ
thymidine uptake would be influenced by any agents
present in testing samples that affect cell growth
and/or cell division. Moreover, the assay takes at least
3 days. Noting that activity of IL-l is reflected by the
degree of IL-I binding to target cells, and that the
binding process would take at most 1 h, the determinatiQn of IL-I activity by measuring the quantity of
IL-I molecules bound to target cells would be a plausible alternative to circumvent those problems mentioned above, although it does not manifest the physiological functions of IL-I like thymocyte proliferation.
Binding of IL-I molecules to target cells has been
measured by counting radioactivities after having 1251_
labeled IL-I react with the target cells (Dower et at.,
1985). An alternative for determining the quantity of
bound IL-I could be measuring fluorescence intensity
after treating with fluorochrome-labeled IL-l, in this
study, IL-I-FITC Flow cytometric analyses of thymocytes bound to IL-I-FITC provided reproducible and
convenient way of measuring the fluorescence intensity. It would be an advantage that flow cytometry
does not produce any hazardous radioactive wastes.
Furthermore, flow cytometry makes it possible to analyze more than one parameter in a single experiment
by employing ligands or antibodies tagged with fluorochromes of different kinds such as phycoerythrin,
texas red and propidium iodide in addition to fluorescem.
In receptor binding assay using IL-l-FITC, the degree of IL-l-FITC binding to its receptor on target
339
cells, expressed in terms of MFI, was proportional to
the quantity of IL-l-FITC added. Determination of
the quantity of unlabeled IL-l molecules in testing
samples was also possible by measuring the degree
of competition of unlabeled IL-l molecules with ILl-FITC molecules for the same receptor. This assay
was not much influenced by the number of cells used
in the assay, whereas thymocyte proliferation assay
was greatly influenced by the number of cells used.
Effect of temperature was quite remarkable in the two
different assays. Thymocyte proliferation assay would
strictly require 37 °C as an incubation temperature
since the thymocytes must be cultured at least 3 days.
Receptor binding assay, on the other hand, which
could be completed in I h, remained successful in
the temperatures above 25 °C up to 45 °C.
Receptor binding assay could also be applied to
the assay 9f IL-l inhibitors provided that the inhibitors be competitive with IL-l for the same receptor.
Urine from febrile patients has been reported to have
strong inhibitory activity against IL-l, although the
tissues that released these protein inhibitors within the
body are not yet clear (Larrick, 1989). It was suggested
that the inhibitor blocks IL-l function in a competitive
manner (Seckinger et aI., 1987; Carter et aI., 1990; Hannum et at., 1990). In this study a protein fraction
isolated from human febrile urine was assayed for
IL-I inhibitory activity. The binding of IL-I-FITC was
inhibited at all the temperatures tested and in a dose
dependent manner by the urine-derived protein fraction. It showed a typical saturation curve with increasing amount of the inhibitor, suggesting that its inhibition was specific for the IL-l receptor. The inhibitor
also blocked IL-l-dependent thymocyte proliferation
(data not shown).
Expression of IL-l receptors on cell surface could
be analyzed by this receptor binding assay. Surface
markers other than IL-I receptors, e.g., epitopes detected by Jlj.lO or Jlld.2 antibodies could be analyzed,
simultaneously. Thymocytes that expressed IL-I receptors appeared also positive for thy-l marker, and they
comprised more than 95% of total cells. Thymocytes
expressing IL-I receptor molecules on their surface
was divided into two groups in terms of Jlld.2 marker
expression - about two thirds of strong positives and
a third of weak positives or negatives. Thus, multiple
analysis of cell surface markers on the thymocytes
or other cell types including IL-l receptors might provide information regarding cellular changes such as
differentiation and activation.
Taken together, this receptor binding assay has advantages over biological assay of IL-l by thymocyte
proliferation or receptor binding assay using radioactive IL-I in several points. Therefore, this assay could
be a convenient alternative for IL-I assays using thymocyte proliferation or radioactive IL-l.
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Interleukin-\ Assay by Flow Cytometry
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