Particle concentration fluorescence immunoassay for measuring interleukin-6 receptor numbers

PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY FOR MEASURING INTERLEUKIN-6 RECEPTOR NUMBERS Zao-Dung Ling,’

Stephen Gillis,’

Liza J. Hart,’

David S. Matheson’,*

Analysis of the number of receptors per cell and the affinity of the ligand/receptor interaction has provided considerable insight into the functioning of numerous cytokines. Interleukin-6 (K-6) is a multifunctional cytokine which may have considerable clinical relevance in inflammatory or immunodeficiency diseases. Using particle concentration fluorescence immunoassay (PCFIA) technology, an assay is described which calculates the receptor number and affinity on small numbers of human cells. Resting B cells are shown to lack IL-6 receptors but activation of B cells induces up to 1,300 receptors per cell, with & of 1 x lo-” to 2 x lo-“M. Other recombinant mediators do not alter the binding of labeled IL-6 to the cells. PCFIA avoids the use of radioactivity and requires very small numbers of cells (2 x lo4 per well). Potential application to the study of regulatory mechanisms and to clinical situations where small samples of blood are available is feasible. Copyright o 1991 by W.B. Saunders Company

Interleukin-6 (IL-6) is a 26,000 molecular weight protein with a multiplicity of functions. It has been characterized as a B-cell differentiation factor and may be an absolute requirement for the ultimate production of immunoglobulin.‘,’ It is a hybridoma growth factor which is the basis of many of the biological assays.3It has also been shown to be one of the major, if not the main, stimulant for the acute phase response and acts as a hepatocyte-stimulating factor.4 It is produced by many cells in the body with the monocyte/ macrophage lineage likely being the most important source. Endothelial cells, fibroblasts, and activated T cells appear also to be able to produce IL-6.’ Many cells in the body have receptors for IL-6. These include T cells, activated B cells, and hepatocytes.’ It is probable that the number of receptors per cell is comparatively low on many cells. Relatively little has been published regarding the factors which regulate the IL-6 receptor on the various cell types. Current assays to assess the number of receptors require a radioactive ligand (IL-6) plus a relatively large number

‘Division of Immunology, Department of Paediatrics, University of British Columbia, Vancouver, B.C., Canada. ZImmunex Research and Development Corporation, Seattle, WA, USA. *To whom correspondence should be addressed at University of British Columbia, Department of Paediatrics, 4480 Oak Street, Vancouver, B.C., V6H 3V4, Canada. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0301-0010$05.00/0 KEY

WORDS:

CYTOKINE,

fluorescein/IL-6

receptor/immunoassay/PCFIA

Vol. 3, No. 1 (January),

1991: pp 17-20

of cells. Particle concentration immunofluorescence assay (PCFIA) offers a technology which can circumvent some of these restrictions. PCFIA is a technique developed by the Pandex Division (Baxter Corporation) that is based upon the quantitative fluorescence emission detectable in specialized fluoricon assay plates. These plates have the configuration of a standard 96-well microtiter plate but have a filter at the bottom of each well (0.22 microns). This filter retains absorbent beads or cells but allows solubilized material to pass through. The technology to perform these assays involves the use of an automated Pandex Screen Machine to perform reagent addition, washes, and fluorescence quantitation.6 Using the PCFIA technology, an assay for characterizing the number of IL-6 receptors and their affinity for IL-6 was established. RESULTS Increasing concentrations of fluorescein isothiocyanate-conjugated recombinant IL-6 (FITC-rIL-6) show a typical hyperbolic curve in terms of bound FITCrIL-6 (Fig. 1). By plotting the information derived from Fig. 1 as a Scatchard plot (Fig. 2) the number of receptors estimated from this technology for the SKW6.4 cells was 4,200 receptors/cell. The affinity binding constant was 10 x 10-l’ M. Tonsillar B cells were incubated with Staphylococcus aureus Cowan Strain 1 (SAC) for a variable length of time and the number of receptors and dissociation constants calculated. As shown in Table 1, resting B 17

18 I Ling et al.

CYTOKINE,

Vol. 3, No. 1 (January 1991: 17-20)

Table 1. IL-6 receptor on SAC-stimulated tonsillar B cells. B lymphocytes were enriched from human tonsillar cells. IL-6 receptor number and I(d were analyzed prior to and after incubation with SAC.

Time (h)

i= iY

0 24 48 72 96

400 300 200

Receptors/cell

% (PM)

0 80 260 630 470

7 11 26 24

100 v

o;,,,,,,,,,,,,,, 0

Figure

1.

5

10

Specific

15

20

binding

25

30

35

40

45

50

FITC-rlL-6

(PM)

of FITC-rIL-6

to SKW6.4

55

60

65

70

cells.

Twenty thousand SKW6.4 cells were incubated with varying amounts of FITC-rIL-6 as shown. The amount of bound FITC-rIL-6 as expressed as molecules/cell is shown on the Y axis. The data demonstrate the typical hyperbolic relationship.

cells have no IL-6 receptors present. This increases to a maximum of approximately 600 receptors/cell after a 72-h incubation with SAC. There is a gradual decrease in the affinity of the IL-6 receptors with time, since the K,, increased approximately fivefold from 7 x 10-l’ to 26 x lo-‘*M. The inability of other recombinant mediators to block the FITC-rIL-6 binding to B9 cells is shown in Fig. 3. The recombinant mediators IL-l, IL-2, IL-4, IL-5, and interferon-y (IFN-y) were used in concentrations up to a lOOO-fold molar excess (100 nM). None of these recombinant mediators inhibited the binding of FITC-rIL-6 to the B9 cells. Only unlabeled rIL-6 was

able to inhibit the binding of the FITC-rIL-6 to the receptor. To determine if the receptor number could be correlated to the response to IL-6, B lymphocytes were cultured with various concentrations of SAC for 3 days, washed, and then exposed to 100 U of IL-6. After incubation for 3 days in culture, the number of IL-6 receptors was determined. Increased SAC concentrations from suboptimal (1:40) to optimal (1:lO) caused an increase in IL-6 receptor number, Furthermore, incubation of these cells for an additional 3 days with 100 U of IL-6 induced IgM secretion. The amount of IgM secreted was correlated with IL-6 receptor number (Fig. 4).

DISCUSSION The results obtained from the PCFIA technique to determine receptor number and affinity are very similar to published results obtained by the standard methods.’ Taga et al. have also shown that resting B cells have no IL-6 receptors expressed but, after 2 days % of BINDING 120

500,

100 80 60

dL-4

dL-5

r-IFN

rlL-6

no inhibitor

INHIBITORS -1 FITC-rlL-6: 0 , 0

I 1000

I 2000

Bound

Figure cells.

2.

Scatchard

analysis

I 3000

,\ 4000

I 5000

Figare

3.

nM

10 nM

0

100 nM

100 pt.4

Inhibition

of rIL-6

binding

to B9 cells.

(molecules/ceil)

of the FITC-m-6

binding

to SKW6.4

The Scatchard analysis of the binding to the SKW6.4 cells as demonstrated in Fig. 1 are shown.

The recombinant mediators shown were added in varying concentrations at the same time as the FITC-rIL-6 to assess their ability to inhibit the binding of FITC-rIL-6 to B9 cells. Clearly, only the rIL-6 was able to inhibit the binding to the cells; no significant inhibition was noted for the other mediators even at concentrations of l,OOO-fold molar excess.

IL-6

RECEPTORS(sites/cell)

IgM(ug/ml)

14007

13500

1200

3000

1000

2500

600

2000

600

1500

400

1000

200

500

0

0 1:lO

DILUTIONS =

Figure

4.

1:40

I:20

RECEPTOR

Correlation

of receptor

OF SAC @@ IgM SECRETION

number

and IgM

secretion.

B lymphocytes enriched from tonsillar cells were incubated with varying concentrations of SAC. An optimal concentration of SAC is recorded as 1. The number of IL-6 receptors was determined after 3 days. Cells were then incubated with 100 U of IL-6 for another 3 days and the amount of IgM secretion was determined. There appears to be some correlation between IL-6 receptor number and functional responsiveness to IL-6.

of SAC activation, the presence of 230 receptors/cell was demonstrated with a Kd of 3.3 x 10-l’ M. Our results indicate very similar numbers of receptors expressed with a maximum being obtained after 3 days of culture with SAC (Table 1). The I(d obtained by our methods indicates a higher affinity of approximately 1 x 10-l’ to 2 x 10-l’ M. The ubiquitous nature of the IL-6 receptor implies it has significant roles to play in vivo. Kishimoto et al. have suggested that IL-6 is necessary for normal immunoglobulin secretion to 0ccur.l If the IL-6 contribution to cell differentiation is as critical as that, diseases may be found which occur due to the impaired production of IL-6 or, potentially, an impaired interaction between IL-6 and its receptor. Our laboratory has evidence suggesting that IL-6 production by peripheral blood mononuclear cells of patients with hypogammaglobulinemia is normal in most cases (Matheson et al., in preparation). Only one patient has been found in our investigations to have an impaired production of a B-cell differentiation factor.’ The ability to further assess the ligand/receptor interaction at the clinical level may provide further information in various diseases such as antibody deficiency syndromes. The PCFIA technology provides distinct advantages over the normal radioactive technique for receptor analysis. Using PCFIA, there is no requirement for radioactivity since the signal is produced by FITC. FITC-labeled proteins are relatively stable and hence there is no decay such as occurs with 1251-labeling. The assay is very rapid, with the above experiments taking approximately 30 min to perform. Perhaps the most important factor is the relatively small number of cells required to perform each assay. Many radioactive

receptor number and affinity by PCFIA

/ 19

assays require l,OOO,OOOcells/tube whereas this technique uses 20,000 cells/well. With this small number, considerably more investigations can be performed on the various cell subpopulations from a reasonably small sample of patient blood. The application of this technique to other mediators is almost certainly feasible. We have recently established a similar assay for the assessment of IL-4 receptors on B cells (data not shown). Some cautionary notes are appropriate with this technique. The determination of the maximum amount of FITC that can be bound by simply “spotting” a small amount of material directly on the Fluoricon plate requires that the material contain only purified ligand and that the unbound FITC is properly removed. Furthermore, nonspecific binding of the FITC-labeled material must be determined and minimized. A major advantage is the ability to use small amounts of cells, hence maintaining a low molar ratio of receptor number to K,, a relationship that enhances the mathematical modeling of receptor/ligand interactions (see reference 11 for an excellent review). The PCFIA technique for assessing the number of IL-6 receptors and their affinity has considerable advantage over the radioactive technique and the results obtained are very similar. It is probable that PCFIA is applicable to other ligand/receptor interactions.

MATERIALS AND METHODS Mediators Recombinant IL-6 (rIL-6) was a generous gift from Immunex Research and Development Corporation, Seattle. A purified fraction of IL-6 was conjugated with fluorescein isothiocyanate (FITC) by previously published methods.8 Labeled rIL-6 was passed through a G25 Sephadex column to remove free FITC, and the recovered material was dialyzed twice against phosphate-buffered saline (PBS). Bovine serum albumin (1%) was added to the FITC-rIL-6 before it was

frozen into aliquots. The mole ratio of IL-6 to FITC was calculated to b 1:2. Other recombinant mediators, IL-l, IL-2, IL-4, IL-5, Corporation.

and IFNq,

were

also provided

by Immunex

Cells The SKW6.4 cell line was purchased from the American Type Culture Collection, Rockville, MD. It is an EpsteinBarr virus-positive human B-cell line which responds to IL-6 by secretion of IgM.9 B9 is a murine B-cell hybridoma which proliferates in the presence of human IL-6 and was kindly provided by Dr. P. Landsdorp, Terry Fox Laboratories, Vancouver, B.C.“’ Human tonsils were minced with scissors, vortexed, and passed through a funnel containing sterile glass wool to remove the solid debris. The recovered cell suspension was used to prepare a B lymphocyte-enriched population by the 2-aminoethylisothiouronium bromide (AET)

20 / Ling et al.

CYTOKINE, Vol. 3, No. 1 (January1991:17-20)

rosetting method.’ The B lymphocytes enriched from the tonsillar cells were cultured with StaphyZococcu.raurez4.r Cowan Strain 1 (SAC) at approximately 10’ organisms/ml (the optimal dilution to induce proliferation of B cells from our laboratory stock is 1:lO). Cells were recovered at various times after the initiation of culture to assessthe IL-6 receptor number.

the British Columbia Health Care Research Foundation. We thank Dr. G. Krystal for reviewing the manuscript and gratefully acknowledge the secretarial assistance of Marian Schaumleffel and the data analysis by Ian Mitchell.

PCFL4 Assayfor IL-6

REFERENCES

The plates were pre-treated with a solution of 1% BSA in PBS, pH 7.0, for 1 h at room temperature. Twenty thousand cells were added to each well of the Baxter Fluoricon assayplate. Cells were incubated with FITC-rIL-6 in PBS, pH 7.0, at various concentrations (0.5 pM to 65 PM) on ice for 20 min. Twenty minutes appeared to be adequate to ensure saturation of the receptors (data not shown). The plates were then inserted into the Pandex Screen Machine where the free FITC-rIL-6 was removed and the cells were automatically washed twice with PBS.The number of relative fluorescence units of the bound FITC-rIL-6 was read by the Pandex Screen Machine. Where indicated, a lo- to lOOO-fold molar excessof unlabeled mediators was added to determine their ability to block binding of the FITC-rIL-6.

1. Muraguchi A, Hirano T, Tang B, Matsuda T, Horii Y, Nakajima K, Kishimoto T (1988) The essential role of B cell stimulating factor 2 (BSF-2/IL-6)for the terminal differentiation of B cells.J ExpMed 167:332-344.

Calculation of the Number of Receptorsof IL-6 Per Cell Relative fluorescence units (RFU) were determined by adding 2 ul of each concentration of the FITC-rIL-6 to the Baxter Fluoricon plate in triplicate. The RFU values were read directly. The number of RFU per molecule was determined by dividing the RFU value by the number of molecules of FITC-rIL-6 added. The total number of molecules of FITC-rIL-6 bound was determined by dividing the total RFU value by the number of RFU per molecule. Finally, the value for total bound FITC-rIL-6 was divided by the number of cells per well to establish the number of receptors on each cell. The I
Acknowledgments This work is supported by grants from the Medical Research Council, the Canadian Arthritis Society, and

2. Kishimoto T, Hirano T (1988) Molecular regulation of B lymphocyte response. Annu Rev Immunol6:485-512. 3. Aarden LA, Groot ER, Schaap OL, Lansdorp PM (1987) Production of hybridoma growth factor by human monocytes.Eur J Immunol17:1411. 4. Gauldie J, Richards C, Hasnish D, Lansdorp P, Baumann M (1987) Interferon p2/B cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Nat1 Acad Sci USA 84:7251-7255. 5. Taga T, Kawanishi Y, Hardy RR, Hirano T, Kishimoto T (1987) Receptors for B cell stimulatory factor 2. Quantitation,

specificity,distributionand regulationof their expression.J ExpMed 166:967-981. 6. Dosch H-M, Leeder S, Cheung R, Lam P, Spielberg S (1988) Realities and future of PCFIA for cellular applications in biological research and clinical medicine. Proceedings of the 1987 Pandex

Symposiumon PCFIA, Baxter,Chicago,p 189. 7. Matheson DS, Green BJ (1987) Defect in production of BCDF-like activity by mononuclear cells from a boy with hypogammaglobulinemia. J Immunol138:2469-2472. 8. The TH, Feltkamp TEW (1970) Conjugation of fluorescein isothiocyanate to antibodies (I) Experiments on conditions of conjugation. Immunology 18:865-870. 9. Saiki 0, Ralph P (1983) Clonal difference in response to T cell replacing factor for IgM secretion and TRF receptors in a human B lymphoblast cell line. Eur J Immunol 13:31-34. 10. Lansdorp PM, Aarden LA, Calafat J, Zeiljemaker WP (1986) A growth-factor dependent B-cell hybridoma. Curr Top Micro and Immunol 132:105-113. 11. Limbird LE (1986) Cell Surface Receptors: A Short Course on Theory and Methods. Kluwer Academic Publishers, New York.