ADONIS
Clin. exp. Immunol. (1991) 85, 307-311
000991049100235Q
Recombinant interferon-gamma inhibits the expression of IL-4 receptors on human lymphocytes K. A. BYRON, G. A. VARIGOS* & A M. WOOTTON Departments of Biochemistry and *Dermatology, Royal Melbourne Hospital, Parkville, Victoria, Australia
(Acceptedfor publication
1
March 1991)
SUMMARY -Interferon gamma (IFN-y) has been shown to inhibit many of the activities of IL-4, including the induction of IgE synthesis and the proliferation of T cell clones. Here we demonstrate that IFN-y is able to inhibit the expression of IL-4 receptors on peripheral blood lymphocytes from both normal healthy donors and from patients with chronic lymphocytic leukaemia. Inhibition was shown to be dose-dependent and did not affect the binding affinity of the receptor as shown by Scatchard analysis. IFN-y was unable to displace labelled IL-4 from its membrane receptor, which demonstrates that IFN-y and IL-4 do not compete for the same membrane binding protein. The ability of IFN-y to down-regulate IL-4 receptors may be important in controlling certain immune responses. Keywords IL-4 interferon-gamma receptors IgE
INTRODUCTION IL-4 is a glycoprotein produced by activated T cells and mast cells (Mosmann & Coffman, 1989). It is known that IL-4 has pleiotropic effects on a number of cell types. For example, IL-4 is able to induce the proliferation of activated B cells, T cells and thymocytes (Zlotnick et al., 1987; Paul, 1989). In addition, it induces the expression of both class II MHC antigens and CD23, the low affinity receptor for IgE (Sarafarti & Delespesse, 1988), and is able to stimulate the production of IgE by peripheral blood lymphocytes (PBL) (Pene et al., 1989). IL-4 is also required for the growth and maturation of mast cells (Paul, 1989). The receptor for IL-4 (IL-4R) was recently cloned from the mouse (Mosley et al., 1989). In humans, the IL-4R has been found on a number of haematopoietic and non-haematopoietic cell types (Park et al., 1987). Cross-linking studies have identified a trimolecular complex consisting of a 65/70-kD doublet and a 1l0-kD protein (Foxwell, Woerly & Ryffel, 1989). It has been demonstrated that both insolubilized anti-IgM antibodies and Staphylococcus aureus Cowan protein (SAC), as well as IL-4 itself, have the ability to up-regulate the expression of IL-4R on lymphocytes (Zuber et al., 1990). Gamma-interferon (IFN-y) has been shown to inhibit many of the activities of IL-4. These include the IL-4 induction of IgE production (Pene et al., 1988) and the IL-4 induced proliferation of some T cell clones (Schmitt et al., 1990). The mechanisms by which IFN-y inhibits the action of IL-4 remain unknown. Correspondence: Dr K. A. Byron, Department of Biochemistry, Royal Melbourne Hospital, Grattan Street, Parkville 3050, Victoria, Australia.
307
Recently it has demonstrated that PBL and allergen-specific T cell clones from patients with allergic disease have a preferential ability to produce IL-4 over IFN-y compared to cells from non-allergic donors (Romagnani et al., 1989: Wierenga et al., 1990). Therefore, the implication of an imbalance in the production of IFN-y and IL-4 in the pathogenesis of allergic disease led us to investigate whether IFN-y may act to inhibit IL4 activities by down-regulating the expression of IL-4R. In this paper we show that IFN-y is able to decrease the binding of iodinated IL-4 to PBL. This decrease in binding was not due to an alteration in the binding affinity of the IL-4R as demonstrated by Scatchard analysis. MATERIALS AND METHODS Cell culture PBL from five normal healthy donors and from three patients with chronic lymphocytic leukaemia (CLL) were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation. The PBL were washed once with phosphate-buffered saline (PBS) and twice with PBS containing 20% fetal calf serum (FCS). For the induction of IgE synthesis, cells were cultured in an enriched Iscove's medium (Flow, UK) containing 50 yg/ml transferrin, 5 pg/ml insulin and 10% FCS (Classen, Levine & Buckley, 1990), at a concentration of 1 x 106 cells/ml. Aliquots of 1 ml were transferred to 12 x 75 mm polystyrene tubes where recombinant IL-4 (Schering-Plough, Bloomfield, USA) was added (6 ng/ml) in the presence or absence of varying concentrations of IFN-y (Genetech, South San Francisco, CA). Cultures were incubated at 370C and 5% CO2 for 10 days. Cyclohexamide
308
K. A. Byron, G. A. Varigos & A. M. Wootton
(50 jMg/ml) was added to control tubes to determine pre-formed or cell-associated IgE. Cells for receptor studies were cultured in RPMI 1640 (CSL, Melbourne, Australia) plus 10% FCS and cultured for 24 h with and without IFN-y. Cell viability was determined by the trypan blue exclusion method. Viabilities remained at > 98% for all experiments.
Determination of IgE Flat-bottomed microtitre plates (Nunc, Roskilde, Denmark) were coated overnight with a polyclonal rabbit anti-human IgE antibody (Dakopatts, Glostrup, Denmark). After the plates were washed with PBS/0-05% Tween, unbound reactive sites were blocked with 0-1% bovine serum albumin (BSA). Standards and cell supernatants were added and incubated at 40C for 3 h, after which a MoAb (Immunotech, France), which recognizes the Ds-2 epitope on human IgE, was added. After 2 h at 40C a rabbit anti-mouse immunoglobulin antibody coupled to peroxidase was added. Colour was developed using 3,3',5,5'teramethylbenzidine-hydrogen peroxide. The reaction was stopped after 30 min with I M phosphoric acid and absorbances read at 450 nm using a Whittaker EIA reader. The standard used (Behring, Marburg, Germany) was calibrated against WHO reference material.
Jodination of IL-4 IL-4 (1 jug) was iodinated with 1 mCi '25Iodine (Amersham, Amersham, UK) using a two-phase radioiodination method (Tejedor & Ballesta, 1982). Labelled IL-4 was separated from free 125Iodine using a Bio-gel P-6DG column (Biorad, Richmond, CA). After the addition of BSA (5 mg/ml), labelled IL-4 was stored at 4°C for up to 4 weeks. Purity and molecular weight determination of the labelled IL-4 was performed by SDSPAGE followed by autoradiography. The maximal binding and specific activity of the labelled IL-4 was determined by methods described by Taga et al., (1987). Binding assay The binding assay was performed by a modification of the method described by Nicola & Metcalf (1985). Cells were washed twice in PBS and resuspended binding medium (RPMI, 20 mm HEPES, 3% BSA and 0-2% NaN3). Cells (3 x 106) were mixed with '251-IL4 with and without a 200-fold excess of unlabelled IL-4 in a final volume of 70 jil and incubated on ice for 4 h with occasional agitation. At the end of incubation, cells were layered onto a 200-pl cushion of silicone oil (Hopkins and Williams, UK) in tapered 400 yl polyethylene tubes and centrifuged at 12 000 g for 90 s (Microfuge, Beckman, USA). Cell pellets were then isolated by cutting the tubes 2-3 mm above the pellet with a scalpel and cell-associated radioactivity was measured on a LKB Gamma Master counter. Specific binding was determined by subtracting the counts of the sample containing a 200-fold excess of unlabelled IL-4 (non-specific
binding). RESULTS Inhibition of IL-4 induced IgE production by IFN-y PBL were cultured in an enriched Iscove's medium with 6 ng/ml IL-4 in the presence or absence of varying concentrations of IFN-y. As shown in Table 1, the exogenous IL-4 added to cultures led to a marked increase in IgE production over control
Table 1. Effect of IFN-y on IL-4-induced IgE production
Addition(s)
Net IgE ng/mI
Medium 6 ng/ml IL-4
< 0-2 15-1 14-0 6-1 2-5
IL-4+ IFN-y (50 U/ml) IL-4+IFN-y (100 U/ml) IL-4+IFN-y (500 U/ml)
kD
97T 4-
66.2 -
42.69
31 _
21 -
14 4
Fig. 1. Autoradiograph of labelled IL-4
as
analysed by SDS-PAGE
(12% gel). The molecular mass (kD) of protein standards are indicated. cultures. The addition of IFN-y inhibited the IL-4 induction of IgE in a dose-dependent manner with maximal inhibition occurring at 500 U/mI. In the following studies this concentration was used to examine the effects of IFN-y on expression of IL-4R.
Radiolabelling of recombinant IL-4 Fractions containing '251-IL-4 were pooled and subsequently analysed for purity by SDS.PAGE. Fig. 1 shows an autoradio-
309
IFN-y inhibits IL-4 receptor expression 0-7
0-6 I\ M
Normal PBL
0 *4 CLL
0*3 20
80 100 40 60 Total binding (% of control)
120 0-2
Fig. 2. '25I-IL-4 binding by PBL cultured in the presence (blank bars) or absence (shaded bars) of 500 U/ml IFN-y. Total counts are expressed as a percentage of the specific counts bound to control cells. (Means + s.e.m. for triplicate assays.)
0*1l
.
0
Bound (receptors/cell ) Fig. 4. Scatchard plot of IL-4R on PBL from a patient with CLL in the presence (crosses) or absence (squares) of IFN-y.
Control
50 U IFN Control
100 U IFN
IFN-y
500 U IFN IL-4
120 Total binding (% of control)
Fig. 3. '25I-IL-4 binding PBL cultured with increasing concentrations of IFN-y for 24 h (shaded bars) or 48 h (open bars). Results are expressed as the means + s.e.m. for triplicate assays.
graph of the pooled fractions, demonstrating the presence of a single band with an approximate molecular weight of 20 kD corresponding to IL-4. The specific activity was calculated to be 3-2 x 10'5 ct/min per millimole. The effect of IFN-y on IL-4 receptors PBL from five normal healthy donors and three CLL patients were cultured for 24 h in the presence and absence of 500 U/ml IFN-y. Fig. 2 shows that IFN-y reduced the binding of labelled
0
20
40 60 80 Total binding (% of control)
100
120
Fig. 5. Competition for IL-4 binding sites. 3 x 106 cells and 1O 000 ct/min of 1251-IL-4 were incubated for 4 h with either 500 U/ml IFN-y or 8 ng/ml IL-4. Results are expressed as the means + s.e.m. of triplicate assays.
IL-4 to PBL compared with the control cells. This effect was with both the normal healthy PBL and those from CLL patients, with the reduction being greater in the latter group, which may reflect differences in cell populations. This reduction of IL-4 binding was examined in more detail by culturing CLL patients' PBL for 24 or 48 h with varying concentrations of IFNy. As demonstrated in Fig. 3, the reduction in binding appears to seen
310
K. A. Byron, G. A. Varigos & A. M. Wootton
be dose dependent with further reductions occurring at 48 h (with the exception of the 500 U dose). Scatchard analysis performed on three separate CLL patients' PBL revealed an average of 210 + 23 IL-4 receptors per cell with a Kd of 55 + 18 pM (mean + s.e.m.). When the cells were incubated for 24 h with IFN-y, the receptor number fell to 1 10 + 50 with no apparent change in the binding affinity, as seen by a representative Scatchard plot from one patient (Fig. 4). Similar results were seen with the PBL from the other two patients (data not shown). Displacement analysis was performed to demonstrate that IFN-y did not compete with IL-4 for its membrane receptor. Figure 5 clearly shows that IFN-y could not displace labelled IL-4 from PBL receptors whereas approximately 85% of '25I-IL-4 was displaced by unlabelled IL4.
DISCUSSION In this paper we have demonstrated that IFN-y inhibits the expression of IL-4R on human PBL. This was shown to occur on PBL from both normal healthy donors and from patients with CLL. Scatchard analysis demonstrated that the downregulation of IL-4R had no effect on binding affinity. The observating that saturation levels of IFN-^y could not displace labelled IL-4 from its membrane receptor indicates that IFN-,, and IL-4 do not compete for the same membrane binding protein. Our data, therefore, suggests a possible relationship between the surface receptors for IL-4 and for IFN-y,. This is not surprising since IFN-y has been shown to inhibit many of the activities of IL-4. In mice, T helper cells can be divided into two distinct subsets (THI or TH2) according to the array of lymphokines they secrete (Mosmann et al., 1986). IFN--y is able to regulate the differential activation of these T cell subsets (Gajewski et al., 1989). In particular, IFN-y which is secreted by THI cells, is able to inhibit IL-4 induced proliferation and expansion of TH2 cells. IFN-y has also been shown to inhibit the IL-4-induced production of IgE, as well as the IL-4-induced expression of CD23 (Pene et al., 1988). The relationship between IL-4 and IFN-y receptors may be similar to that demonstrated by IL-3 and GM-CSF which appear to have a reciprocal inhibition of binding to eosinophils (Lopez et al., 1989). Further studies are currently underway to determine whether IFN-j acts directly to down-regulate the translation of the IL-4 receptor protein or whether it effects the process of internalization. Because PBL from our patients with CLL comprised more than 95% B cells, it is likely that IFN-y is able to inhibit the expression of IL-4R on B cells. Also, it is now apparent that IL-4 acts on cells which have already been activated, suggesting that the process of activation leads to an increased expression of membrane receptors for IL-4. Such a process has been demonstrated in vitro, where SAC and insolubilized anti-IgM antibodies were able both to activate B cells and to increase the number of IL-4R (Foxwell et al., 1989). Several groups (Romagnani et al., 1989; Wierenga et al., 1990) have demonstrated a decreased production of IFN-y from both PBL and allergen-specific T cell clones in patients with allergic disease. In these patients, the increased expression of CD23 (a marker of B cell activation) and the over-production of IgE (Bujanowski-Weber et al., 1989) may be due to a loss of the ability to control IL-4R expression, by IFN-y which leads to a
continual B cell activation. The ability to inhibit or downregulate the expression of IL-4R may therefore demonstrate a natural controlling mechanism to suppress the activities of IL-4 in certain immune responses. The failure of this mechanism may be of particular importance in the development and maintenance of allergic disease, where an imbalance between IL-4 and IFN-j is thought to play a central role in the pathogenesis. Therefore, further elucidation of this mechanism may be of great clinical benefit.
ACKNOWLEDGMENTS This research was partly supported by grants from the Asthma Foundation of Victoria and the Victor Hurley Medical Research Fund of the Royal Melbourne Hospital. We are grateful for the helpful discussion and review of the manuscript by A. Boyd and S. Ratnaike.
REFERENCES BUJANOWSKI-WEBER, J., BRINGS, B., KNOLLER, I., PFEIL, T. & KONIG, W. (1989) Expression of low-affinity receptor for IgE (FcERII, CD23) and IgE-BF (soluble CD23) release by lymphoblastoid B-cell line RPMI-8866 and human peripheral lymphocytes of normal and atopic donors. Irnmunologi', 66, 505. CLASSEN, J.L., LEVINE, A.D. & BUCKLEY, R.H. (1990) A cell culture system that enhances mononuclear cell IgE synthesis induced by recombinant human interleukin 4. J. immunol. Methods, 126, 213. FOXWELL, B., WOERLY, G. & RYFFEL, B. (1989) Identification of interleukin 4 receptor-associated proteins and expression of both high and low affinity binding on human lymphoid cells. Eur. J. Immunol. 19, 1637. GAJEWSKI, T.F., SCHELL, S.R., NAU, G. & FITCH, F.W. (1989) Regulation of T-cell activation: differences among T-cell subsets. Immunol. Rev. 111, 79. LOPEZ, A.F., EGLINGTON, J.M., GILLIS, D., PARK, L.S., CLARK, S. & VADAS, M.A. (1989) Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony stimulating factor to human eosinophils. Proc. natl Acad. Sci. USA, 86, 7022. MOSLEY, B., BECKMANN, P., MARCH, C.J., IDYERDA, R., GIMPEL, S.D., VANDENBOS, T., FRIEND D., ALPERT, A., ANDERSON, D., JACKSON, D., WIGNALL, J.M., SMITH, C., GALLIS, B., SIMS, J.E., URDAL, D., WIDMER, M.B., COSMAN, D. & PARK, L. S. (1989) The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane forms. Cell, 9, 335. MOSMANN, T.R., CHERWINSKI, H., BOND, M.W., GIEDLIN, M.A. & COFFMAN, R.L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348. MOSMANN, T.R. & COFFMAN, R.L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion leads to different functional properties. Annu. Rev. Immunol. 7, 145. NICOLA, N.A. & METCALF, D. (1985) Binding of '251-labeled granulocyte colony stimulating factor to normal murine hemopoietic cells. J. cell. Phvysiol. 124, 313. PARK, L.S., FRIEND, D., SASSENFELD, H.M. & URDAL, D.L. (1987) Characterization of the human B cell stimulatory factor I receptor. J. exp. Med. 166, 476. PAUL, W.E. (1989) Pleiotropy and redundency: T cell derived lymphokines in the immune response. Cell, 57, 521. PENE, J., CHRETIEN, I., RouSSET, F., BRIERE, F., BONNEFOY, J.Y. & DE VRIES, J.E. (1989) Modulation of IL4 induced human IgE production in-vitro by IFN-gamma and ILS. The role of soluble CD23. J. cell. Biochem. 39, 253. PENE, J., RouSSET, F., BRIERE, F., CHRETIEN, 1., BONNEFOY, J., SPITS, H., YOKOTA, T., ARAI, N., ARAI, K., BANCHEREAU, J. & DE VRIES, J.
IFN-y inhibits IL-4 receptor expression (1988) IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2. Proc. natl Acad. Sci. USA, 85, 6880. ROMAGNANI, S., DEL PRETE, G., MAGGI, E., PARRONCHI, P., TIRI, A., MACCHIA, D., GIUDIZI, M.G., ALMERIGOGNA, F. & Ricci, M. (1989) Role of interleukins in induction and regulation of human IgE synthesis. Clin. Immunol. Immunopathol. 50, S13. SARAFATI, M. & DELESPESSE, G. (1988) Possible role of human lymphocyte receptor for IgE (CD23) or its soluble fragments in the invitro synthesis of human IgE. J. Immunol. 141, 2195. SCHMITT, E., VAN BRANDWIJK, R., FISCHER, H.G. & RUDE, E. (1990) Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors. Eur. J. Immunol. 20, 1709. TAGA, T., KAWANISHI, Y., HARDY, R., HIRANO, T. & KISHIMOTO, T. (1987) Receptors for B cell stimulatory factor 2: quantitation
311
specificity, distribution and regulation of their expression. J. exp. Med. 166, 967. TEJEDOR, F. & BALLESTA, J.P.G. (1982) lodination of biological samples without loss of functional activity. Anal. Biochem. 127, 143. WIERENGA, E.A., SNOEK, M., DE GROOT, C., CHRETIEN, I., Bos, J.D., JANSEN, H.M. & KAPSENBERG, M.L. (1990) Evidence for the compartmentalization of functional subsets of CD4+ lymphocytes in atopic patients. J. Immunol. 144, 4651. ZLOTNICK, A., RANSOM, J., FRANK, G., FISCHER, M. & HOWARD, M. (1987) Interleukin 4 is a growth factor for activated thymocytes: possible role in T cell ontogony. Proc. natl Acad. Sci. USA, 84, 3856. ZUBER, C., GALIZZI, J.P., VALLE, A., HARADA, N., HOWARD, M. & BANCHEREAU, J. (1990) Interleukin 4 receptors in normal human B lymphocytes: characterization and regulation. Eur. J. Immunol. 20, 551.
Clin. exp. Immunol. (1991) 85, 307-311
000991049100235Q
Recombinant interferon-gamma inhibits the expression of IL-4 receptors on human lymphocytes K. A. BYRON, G. A. VARIGOS* & A M. WOOTTON Departments of Biochemistry and *Dermatology, Royal Melbourne Hospital, Parkville, Victoria, Australia
(Acceptedfor publication
1
March 1991)
SUMMARY -Interferon gamma (IFN-y) has been shown to inhibit many of the activities of IL-4, including the induction of IgE synthesis and the proliferation of T cell clones. Here we demonstrate that IFN-y is able to inhibit the expression of IL-4 receptors on peripheral blood lymphocytes from both normal healthy donors and from patients with chronic lymphocytic leukaemia. Inhibition was shown to be dose-dependent and did not affect the binding affinity of the receptor as shown by Scatchard analysis. IFN-y was unable to displace labelled IL-4 from its membrane receptor, which demonstrates that IFN-y and IL-4 do not compete for the same membrane binding protein. The ability of IFN-y to down-regulate IL-4 receptors may be important in controlling certain immune responses. Keywords IL-4 interferon-gamma receptors IgE
INTRODUCTION IL-4 is a glycoprotein produced by activated T cells and mast cells (Mosmann & Coffman, 1989). It is known that IL-4 has pleiotropic effects on a number of cell types. For example, IL-4 is able to induce the proliferation of activated B cells, T cells and thymocytes (Zlotnick et al., 1987; Paul, 1989). In addition, it induces the expression of both class II MHC antigens and CD23, the low affinity receptor for IgE (Sarafarti & Delespesse, 1988), and is able to stimulate the production of IgE by peripheral blood lymphocytes (PBL) (Pene et al., 1989). IL-4 is also required for the growth and maturation of mast cells (Paul, 1989). The receptor for IL-4 (IL-4R) was recently cloned from the mouse (Mosley et al., 1989). In humans, the IL-4R has been found on a number of haematopoietic and non-haematopoietic cell types (Park et al., 1987). Cross-linking studies have identified a trimolecular complex consisting of a 65/70-kD doublet and a 1l0-kD protein (Foxwell, Woerly & Ryffel, 1989). It has been demonstrated that both insolubilized anti-IgM antibodies and Staphylococcus aureus Cowan protein (SAC), as well as IL-4 itself, have the ability to up-regulate the expression of IL-4R on lymphocytes (Zuber et al., 1990). Gamma-interferon (IFN-y) has been shown to inhibit many of the activities of IL-4. These include the IL-4 induction of IgE production (Pene et al., 1988) and the IL-4 induced proliferation of some T cell clones (Schmitt et al., 1990). The mechanisms by which IFN-y inhibits the action of IL-4 remain unknown. Correspondence: Dr K. A. Byron, Department of Biochemistry, Royal Melbourne Hospital, Grattan Street, Parkville 3050, Victoria, Australia.
307
Recently it has demonstrated that PBL and allergen-specific T cell clones from patients with allergic disease have a preferential ability to produce IL-4 over IFN-y compared to cells from non-allergic donors (Romagnani et al., 1989: Wierenga et al., 1990). Therefore, the implication of an imbalance in the production of IFN-y and IL-4 in the pathogenesis of allergic disease led us to investigate whether IFN-y may act to inhibit IL4 activities by down-regulating the expression of IL-4R. In this paper we show that IFN-y is able to decrease the binding of iodinated IL-4 to PBL. This decrease in binding was not due to an alteration in the binding affinity of the IL-4R as demonstrated by Scatchard analysis. MATERIALS AND METHODS Cell culture PBL from five normal healthy donors and from three patients with chronic lymphocytic leukaemia (CLL) were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient centrifugation. The PBL were washed once with phosphate-buffered saline (PBS) and twice with PBS containing 20% fetal calf serum (FCS). For the induction of IgE synthesis, cells were cultured in an enriched Iscove's medium (Flow, UK) containing 50 yg/ml transferrin, 5 pg/ml insulin and 10% FCS (Classen, Levine & Buckley, 1990), at a concentration of 1 x 106 cells/ml. Aliquots of 1 ml were transferred to 12 x 75 mm polystyrene tubes where recombinant IL-4 (Schering-Plough, Bloomfield, USA) was added (6 ng/ml) in the presence or absence of varying concentrations of IFN-y (Genetech, South San Francisco, CA). Cultures were incubated at 370C and 5% CO2 for 10 days. Cyclohexamide
308
K. A. Byron, G. A. Varigos & A. M. Wootton
(50 jMg/ml) was added to control tubes to determine pre-formed or cell-associated IgE. Cells for receptor studies were cultured in RPMI 1640 (CSL, Melbourne, Australia) plus 10% FCS and cultured for 24 h with and without IFN-y. Cell viability was determined by the trypan blue exclusion method. Viabilities remained at > 98% for all experiments.
Determination of IgE Flat-bottomed microtitre plates (Nunc, Roskilde, Denmark) were coated overnight with a polyclonal rabbit anti-human IgE antibody (Dakopatts, Glostrup, Denmark). After the plates were washed with PBS/0-05% Tween, unbound reactive sites were blocked with 0-1% bovine serum albumin (BSA). Standards and cell supernatants were added and incubated at 40C for 3 h, after which a MoAb (Immunotech, France), which recognizes the Ds-2 epitope on human IgE, was added. After 2 h at 40C a rabbit anti-mouse immunoglobulin antibody coupled to peroxidase was added. Colour was developed using 3,3',5,5'teramethylbenzidine-hydrogen peroxide. The reaction was stopped after 30 min with I M phosphoric acid and absorbances read at 450 nm using a Whittaker EIA reader. The standard used (Behring, Marburg, Germany) was calibrated against WHO reference material.
Jodination of IL-4 IL-4 (1 jug) was iodinated with 1 mCi '25Iodine (Amersham, Amersham, UK) using a two-phase radioiodination method (Tejedor & Ballesta, 1982). Labelled IL-4 was separated from free 125Iodine using a Bio-gel P-6DG column (Biorad, Richmond, CA). After the addition of BSA (5 mg/ml), labelled IL-4 was stored at 4°C for up to 4 weeks. Purity and molecular weight determination of the labelled IL-4 was performed by SDSPAGE followed by autoradiography. The maximal binding and specific activity of the labelled IL-4 was determined by methods described by Taga et al., (1987). Binding assay The binding assay was performed by a modification of the method described by Nicola & Metcalf (1985). Cells were washed twice in PBS and resuspended binding medium (RPMI, 20 mm HEPES, 3% BSA and 0-2% NaN3). Cells (3 x 106) were mixed with '251-IL4 with and without a 200-fold excess of unlabelled IL-4 in a final volume of 70 jil and incubated on ice for 4 h with occasional agitation. At the end of incubation, cells were layered onto a 200-pl cushion of silicone oil (Hopkins and Williams, UK) in tapered 400 yl polyethylene tubes and centrifuged at 12 000 g for 90 s (Microfuge, Beckman, USA). Cell pellets were then isolated by cutting the tubes 2-3 mm above the pellet with a scalpel and cell-associated radioactivity was measured on a LKB Gamma Master counter. Specific binding was determined by subtracting the counts of the sample containing a 200-fold excess of unlabelled IL-4 (non-specific
binding). RESULTS Inhibition of IL-4 induced IgE production by IFN-y PBL were cultured in an enriched Iscove's medium with 6 ng/ml IL-4 in the presence or absence of varying concentrations of IFN-y. As shown in Table 1, the exogenous IL-4 added to cultures led to a marked increase in IgE production over control
Table 1. Effect of IFN-y on IL-4-induced IgE production
Addition(s)
Net IgE ng/mI
Medium 6 ng/ml IL-4
< 0-2 15-1 14-0 6-1 2-5
IL-4+ IFN-y (50 U/ml) IL-4+IFN-y (100 U/ml) IL-4+IFN-y (500 U/ml)
kD
97T 4-
66.2 -
42.69
31 _
21 -
14 4
Fig. 1. Autoradiograph of labelled IL-4
as
analysed by SDS-PAGE
(12% gel). The molecular mass (kD) of protein standards are indicated. cultures. The addition of IFN-y inhibited the IL-4 induction of IgE in a dose-dependent manner with maximal inhibition occurring at 500 U/mI. In the following studies this concentration was used to examine the effects of IFN-y on expression of IL-4R.
Radiolabelling of recombinant IL-4 Fractions containing '251-IL-4 were pooled and subsequently analysed for purity by SDS.PAGE. Fig. 1 shows an autoradio-
309
IFN-y inhibits IL-4 receptor expression 0-7
0-6 I\ M
Normal PBL
0 *4 CLL
0*3 20
80 100 40 60 Total binding (% of control)
120 0-2
Fig. 2. '25I-IL-4 binding by PBL cultured in the presence (blank bars) or absence (shaded bars) of 500 U/ml IFN-y. Total counts are expressed as a percentage of the specific counts bound to control cells. (Means + s.e.m. for triplicate assays.)
0*1l
.
0
Bound (receptors/cell ) Fig. 4. Scatchard plot of IL-4R on PBL from a patient with CLL in the presence (crosses) or absence (squares) of IFN-y.
Control
50 U IFN Control
100 U IFN
IFN-y
500 U IFN IL-4
120 Total binding (% of control)
Fig. 3. '25I-IL-4 binding PBL cultured with increasing concentrations of IFN-y for 24 h (shaded bars) or 48 h (open bars). Results are expressed as the means + s.e.m. for triplicate assays.
graph of the pooled fractions, demonstrating the presence of a single band with an approximate molecular weight of 20 kD corresponding to IL-4. The specific activity was calculated to be 3-2 x 10'5 ct/min per millimole. The effect of IFN-y on IL-4 receptors PBL from five normal healthy donors and three CLL patients were cultured for 24 h in the presence and absence of 500 U/ml IFN-y. Fig. 2 shows that IFN-y reduced the binding of labelled
0
20
40 60 80 Total binding (% of control)
100
120
Fig. 5. Competition for IL-4 binding sites. 3 x 106 cells and 1O 000 ct/min of 1251-IL-4 were incubated for 4 h with either 500 U/ml IFN-y or 8 ng/ml IL-4. Results are expressed as the means + s.e.m. of triplicate assays.
IL-4 to PBL compared with the control cells. This effect was with both the normal healthy PBL and those from CLL patients, with the reduction being greater in the latter group, which may reflect differences in cell populations. This reduction of IL-4 binding was examined in more detail by culturing CLL patients' PBL for 24 or 48 h with varying concentrations of IFNy. As demonstrated in Fig. 3, the reduction in binding appears to seen
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K. A. Byron, G. A. Varigos & A. M. Wootton
be dose dependent with further reductions occurring at 48 h (with the exception of the 500 U dose). Scatchard analysis performed on three separate CLL patients' PBL revealed an average of 210 + 23 IL-4 receptors per cell with a Kd of 55 + 18 pM (mean + s.e.m.). When the cells were incubated for 24 h with IFN-y, the receptor number fell to 1 10 + 50 with no apparent change in the binding affinity, as seen by a representative Scatchard plot from one patient (Fig. 4). Similar results were seen with the PBL from the other two patients (data not shown). Displacement analysis was performed to demonstrate that IFN-y did not compete with IL-4 for its membrane receptor. Figure 5 clearly shows that IFN-y could not displace labelled IL-4 from PBL receptors whereas approximately 85% of '25I-IL-4 was displaced by unlabelled IL4.
DISCUSSION In this paper we have demonstrated that IFN-y inhibits the expression of IL-4R on human PBL. This was shown to occur on PBL from both normal healthy donors and from patients with CLL. Scatchard analysis demonstrated that the downregulation of IL-4R had no effect on binding affinity. The observating that saturation levels of IFN-^y could not displace labelled IL-4 from its membrane receptor indicates that IFN-,, and IL-4 do not compete for the same membrane binding protein. Our data, therefore, suggests a possible relationship between the surface receptors for IL-4 and for IFN-y,. This is not surprising since IFN-y has been shown to inhibit many of the activities of IL-4. In mice, T helper cells can be divided into two distinct subsets (THI or TH2) according to the array of lymphokines they secrete (Mosmann et al., 1986). IFN--y is able to regulate the differential activation of these T cell subsets (Gajewski et al., 1989). In particular, IFN-y which is secreted by THI cells, is able to inhibit IL-4 induced proliferation and expansion of TH2 cells. IFN-y has also been shown to inhibit the IL-4-induced production of IgE, as well as the IL-4-induced expression of CD23 (Pene et al., 1988). The relationship between IL-4 and IFN-y receptors may be similar to that demonstrated by IL-3 and GM-CSF which appear to have a reciprocal inhibition of binding to eosinophils (Lopez et al., 1989). Further studies are currently underway to determine whether IFN-j acts directly to down-regulate the translation of the IL-4 receptor protein or whether it effects the process of internalization. Because PBL from our patients with CLL comprised more than 95% B cells, it is likely that IFN-y is able to inhibit the expression of IL-4R on B cells. Also, it is now apparent that IL-4 acts on cells which have already been activated, suggesting that the process of activation leads to an increased expression of membrane receptors for IL-4. Such a process has been demonstrated in vitro, where SAC and insolubilized anti-IgM antibodies were able both to activate B cells and to increase the number of IL-4R (Foxwell et al., 1989). Several groups (Romagnani et al., 1989; Wierenga et al., 1990) have demonstrated a decreased production of IFN-y from both PBL and allergen-specific T cell clones in patients with allergic disease. In these patients, the increased expression of CD23 (a marker of B cell activation) and the over-production of IgE (Bujanowski-Weber et al., 1989) may be due to a loss of the ability to control IL-4R expression, by IFN-y which leads to a
continual B cell activation. The ability to inhibit or downregulate the expression of IL-4R may therefore demonstrate a natural controlling mechanism to suppress the activities of IL-4 in certain immune responses. The failure of this mechanism may be of particular importance in the development and maintenance of allergic disease, where an imbalance between IL-4 and IFN-j is thought to play a central role in the pathogenesis. Therefore, further elucidation of this mechanism may be of great clinical benefit.
ACKNOWLEDGMENTS This research was partly supported by grants from the Asthma Foundation of Victoria and the Victor Hurley Medical Research Fund of the Royal Melbourne Hospital. We are grateful for the helpful discussion and review of the manuscript by A. Boyd and S. Ratnaike.
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