Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor

British Journal of Haematology, 1993. 85, 42-49

Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor

ASIM K H W A J A , JULIA CARVER, H. MARK JONES A N D DAVID C . LINCH Department of Haematology, University College London Medical School

Received 13 January 1993; accepted for publication 15 March 1993

Summary. The GM-CSF receptor (GM-CSFR) is composed oft( and subunits. Surface expression of the ct chain alone leads to low affinity GM-CSF binding and of both subunits to high affinity binding: the b chain is required for transducing a proliferative signal. Studies of GM-CSFR expression have concentrated largely on static events occurring under conditions of binding equilibrium. We have examined the dynamic regulation of high and low affinity GM-CSFR expression in neutrophils (1100f200 R/cell, KD 50f 15 p ~ ) and a GM- CSF dependent human leukaemic cell line, TF-l(2000 k 4 5 0 R/cell KD 1 5 k 5 PM) and 8 6 0 0 f 1 1 5 0 R/cell KD 1.8f0.3 nM). The addition of GM-CSF to TF-1 cells (350 PM, 4 h at 3 7 T ) caused a reduction in subsequent binding of lL51-GM- CSF at low ligand concentration (100 PM) (following a low pH wash to remove surface bound ligand) to 1 6 f 4 % and a reduction in binding at high ligand concentration (2 nM lZ4I- GM-CSF) to 36 f 9% of control. Scatchard analysis showed complete down-regulation of high affinity GM-CSFR and a significant reduction in low affinity GM-CSFR. In neutrophils, concentration-response curves of ligand induced receptor

down-regulation at 3 7°C showed that observed down- modulation was more than 10-fold greater than predicted by static equilibrium binding data and correlated closely with GM-CSF priming of the neutrophil respiratory burst. The addition of IL-3 to TF-1 cells at 3 7°C reduced 100 PM lL5I-GM- CSF binding to 1 8 f 4 % and 2 nM lL51-GM-CSF binding to 46 & 5% of control. TF-1 cells, but not neutrophils, were able to re-express GM-CSFR following removal of GM-CSF from medium. TF-1 proliferation assays showed that pulsed GM- CSF (0 .35 -3 .5 nM) for up to 4 h did not cause a significant increase in 3H-thymidine incorporation which required the continued presence of GM-CSF (control 2875 i 208 cpm, pulsed GM-CSF 5 ng/ml49 72 f 1344. continuous GM-CSF 5 ng/ml 17249 f2982). Therefore, proliferation of TF-1 cells required the continued presence of GM-CSF at a time when there was no detectable surface high affinity GM-CSFR. This shows that signal transduction can take place via the GM- CSFR at a time when there is no detectable high affinity GM- CSF binding and introduces a further layer of complexity in the analysis of GM-CSF receptor function.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the production of neutrophil and eosi- nophil granulocytes and monocyte/macrophages both in vitro and in vivo (Metcalf, 1991). In addition, it can enhance the function of mature cells of these lineages (Metcalf, 1991). Binding of GM-CSF to specific surface receptors on target cells is the initial step in the generation of subsequent biological responses. The structure of the GM-CSF receptor (GM-CSFR) has been elucidated and shown to belong to the haemopoietin receptor family (Bazan, 1990; Gearing et a/. 1989; Hayashida et al, 1990). The GM-CSFR is a heterodimer and consists of t(

Correspondence: Dr A. Khwaja. Department of Haematology, University College Medical School, 98 Chenies Mews, London WClE 6HX.

and chains of molecular size approximately 80 and 130 kD respectively. Surface expression of the ct (ligand binding) chain alone leads to low affinity binding (KD 1-10 n M ) and of both chains to formation of a high affinity receptor ( K O 10- 100 PM). Cells may express low affinity receptor alone, high affinity receptor alone or both, although all haemopoietic cells so far described that express GM-CSFR have at least a proportion of high affinity receptors (Nicola, 1991).

Several investigators have examined the respective roles of the ct and b receptor subunits in transducing a proliferative signal (Metcalf et al, 1990: Kitamura et 01. 1991b; Sakamaki et al, 1992). The intracytoplasmic region of the @ chain has been shown to be essential for signalling and the cytoplasmic domain of the t( subunit to be involved in, but not essential for, signal transduction (Sakamaki et nl, 1992). At 37°C.

42

Modulation of GM-CSF Receptor Expression 43

ligand under these conditions (35% at 3 h), they were shown to give maximal binding. Non-specific binding was measured by incubation in the presence of >100-fold excess of unlabelled GM-CSF. Cell suspensions were layered onto a cushion of chilled FBS, centrifuged at 13000 rpm in a desktop microcentrifuge, snap frozen in liquid nitrogen and the cell pellet cut and counted. Binding data were analysed by the EBDA and LIGAND programs (Biosoft, Cambridge, U.K.).

To assess residual surface GM-CSF receptors following treatment with ligand, any remaining bound molecules were removed by a low pH wash (Cannistra et al, 1990). Control cells underwent the identical procedure. Following centrifu- gation, the cell pellet was resuspended in ice-cold PBS, pH 3. for 2 min, topped up with an excess of binding buffer and washed a further three times in binding buffer. In pilot experiments this process did not affect significantly subsequent binding of 1251-GM-CSF ( > 9 5% binding compared with control neutrophils or TF-1 cells) and was shown to remove over 90% of surface bound GM-CSF. Parallel incubations were carried out in the presence of a 100-fold excess of unlabelled GM-CSF to control for non-specific binding.

Re-expression of GM-CSFR in neutrophils and TF-I cells was examined following 2 h (neutrophils) or overnight (TF-1) ligand induced down-regulation (50 ng/ml, 3 7°C). Residual GM-CSF was removed by four large volume washes; TF-1 were resuspended in erythropoietin (2 IU/ml) to maintain viability. Subsequent binding with lL51-GM-CSF was carried out at room temperature for 3 h (neutrophils) or 4°C for 16 h (TF-1). Although, as stated above, internalization of 1251-GM- CSF was observed under these conditions in neutrophils, this did not affect the assay as these counts remained cell- associated.

Measurement of neutrophil respiratory burst activity. Intra- cellular hydrogen peroxide production was measured as previously described (Jaswon et al, 1990). In brief, freshly drawn, whole blood was incubated with 100 p~ DCF-DA for 15 min at 37OC. Fmlp at 10Vh M was then added for 15 min at 3 7OC. The reaction was stopped by placing the samples on ice. Following lysis of red cells, analysis was carried out by flow cytometry using a Coulter (Luton, U.K.) Epics-C machine, neutrophils being selectively gated by virtue of their light- scattering properties.

Proliferation of TF-I cells. TF-1 cells were washed three times to remove GM-CSF from the growth medium and resuspended in RPMI 1640 plus 10% FBS alone for 18 h. GM- CSF was then added at either 5 or 50 ng/ml for up to 4 h. Cells were then washed four times in large volumes of medium and resuspended in medium +neutralizing anti-GM-CSF antibody (pulsed GM-CSF) or in GM-CSF at 5 ng/ml (continuous GM-CSF). Control cells that were maintained starved but underwent the same washing steps were also resuspended in medium+ antibody to control for any non-specific effects. 104 cells/well (quadruplicate points) were incubated overnight and each well was then pulsed with 18.5 kBq 3H-thymidine for 4 h. Viable cell counts were also carried out at this stage.

RESULTS Neutrophil and TF-I GM-CSF receptor expression Scatchard analysis of equilibrium binding data shows that

binding of GM-CSF to high affinity receptors on neutrophils is followed by rapid internalization of the ligand-receptor complex (Cannistra et al, 1990). This process probably serves to down-regulate subsequent responses to GM-CSF as inhibi- tion of receptor internalization leads to increased functional effects of GM-CSF (Khwaja et al, 1990). In contrast to the rapid internalization of the high affinity GM-CSFR, Metcalf has shown that murine cells transfected with the human r chain alone bind GM-CSF with low affinity and internalize bound ligand slowly.

To date, studies of human GM-CSFR kinetics have concentrated on static events occurring under conditions of binding equilibrium with relatively little information on the dynamic regulation ofreceptor expression (Nicola, 1991). In this study we have examined several aspects of GM-CSFR expression at 3 7°C. including the rate and degree of internalization and re- expression of both high and low affinity forms, the influence of occupancy by ligand on internalization of the low affinity receptor, and the relationship between the dynamic regulation of GM-CSFR expression and the biological effects of GM- CSF.

MATERIALS AND METHODS

Materials. '151-GM-CSF (22.2-44.4 TBq/mmol) and %- thymidine ( 1. I 1 TBqlmmol) were obtained from Amersham (Aylesbury, U.K.). Unlabelled recombinant human (rh) GM- CSF produced in E. coli was provided by Behringwerke (Marburg, Germany) and rhIL-3 (produced in E. coli) was provided by Sandoz (Frimley Park, U.K.). Dichloro-fluores- cein diacetate (DCF-DA, Molecular Probes, Oregon, U.S.A.), formyl-met-leu-phe (f MLP) and 12-0-tetradecanoylphorbol 13-acetate (TPA) (both Sigma, Poole, U.K.) were dissolved in DMSO at stock concentrations of 100 mM, 50 mg/ml and 10 mg/ml respectively, stored at -2O"C, and diluted in RPMT (Gibco, Paisley, U.K.) on the day of use. A sheep polyclonal neutralizing antibody against recombinant human GM-CSF was kindly donated by Dr R. Thorpe (National Institute for Biological Standards and Control, South Mimms, U.K.).

Cell lines and purijcution of neutrophils. Neutrophils were prepared from peripheral blood of normal laboratory person- nel by dextran sedimentation and gradient density centrifu- gation as previously described (Khwaja et al, 1990). The human leukaemia cell line TF-1 (Kitamura et al, 1989) was routinely maintained in RPMI 1640 with 10% fetal bovine serum (FBS) and 5 ng/ml GM-CSF.

Binding assays. The specific radioactivity of 1251-GM-CSF was determined by self-displacement analysis after correction for maximal binding capacity (Calvo et al, 198 3). Equilibrium binding assays for Scatchard analysis were carried out with cells incubated in RPMI 1640 with 2 5 mM Hepes and 2% FBS (binding buffer). TF-I cells were washed three times to remove GM-CSF from the medium and resuspended in 2 ItJ/ ml erythropoietin (Boehringer Mannheim, Lewes, U.K.) for 24 h prior to analysis. Binding was carried out at 4°C for 16 h, conditions previously determined to give maximum binding (data not shown). Neutrophils were incubated at room temperature for 3 h as binding at 4°C was affected by cell aggregation. Although there was some internalization of

44 Asim Khwaja et a1

GM-CSF K] 0.40

0.30

0.20

0.10

0.00 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0

cpm bound

Fig 1. Scatchard transformation of '251-GM-CSF binding to TF-1 cells (lO'/point). Cells were incubated with IL-3 (5 ngiml), GM-CSF (5 ng/ ml) or medium alone for 4 h at 3 7°C and then subjected to a low pH wash to remove any surface receptor-hound ligand (see Methods). Incubation was then carried out with varying concentrations of l2jI- GM-CSF at 4'C for 18 h to assess residual GM-CSF receptor expression ( 100-fold molar excess unlabelled GM-CSF was used to control for non-specific binding). Analysis of the results of this experiment show control cells 2 100 R of 20 PM and 8 100 R 1 .5 nM, IL-3 treated 1 1 80 Rice11 1.1 nM, GM-CSF treated 400 R/cell KD 0.8 IIM.

0.20 Ak

E 0 o.loi 0

0 1000 2000 3000 4 0 0 0 5000 6 0 0 0 7000 cpm bound

Fig 2. Representative Scatchard transformation of 12'I-GM-CSF equilibrium binding to neutrophils. One class of receptor was detected with mean KD S o h 1 5 PM, 1100f200 receptors/cell (n=4).

TF-1 cells that have been switched to medium containing epo for 24 h express 2000f450 high affinity (KL, 1 5 f 5 PM) and 8600f 11 50 low affinity (& 1.8 f . 3 nM) GM-CSF receptors (GM-CSFR) per cell (Fig 1). Neutrophils express only one class of high affinity GM-CSFR (KD SO f I 5 PM) with 1100 f 200 receptors per cell ( n = 4 ) (Fig 2).

GM-CSFR expression after addition of ligand Assessment of high and low affinity GM-CSFR expression on TF-1 cells hearing both classes of receptor was carried out by

0 GM-CSF 5 ng/ml GM-CSF 50 ng/ml

.-

u 30 c 4 0 1

4 1 8

time (hours)

Fig 3(a). The effect of incubation with GM-CSF at 37OC on the subsequent binding of 100 PM "'I-GM-CSF binding to TF-1 cells. Cells were incubated with GM-CSF 5 ng/ml (350 PM) or 50 ng/ml (3.5 nM) at 3 7°C subjected to a low pH wash to remove any surface receptor-bound ligand and incubated with 100 PM "'I-GM-CSF for 18 h at 4OC (n= 3). Under baseline conditions, 80% of the cpm hound at this concentration of 'L51-GM-CSF are attributable to binding to the high affinity receptor and the remainder to the low affinity GM- CSFR.

GM-CSF 50 ngiml

T

T

4 1 8

time (hours)

Fig 3(b). The effect of incubation with GM-CSF at 37OC on the subsequent binding of 2 n M '"I-GM-CSF binding to TF-1 cells. Cells were incubated with GM-CSF 5 ng/ml(350 PM) or 50 ng/ml(3.5 nM)

at 3 7% subjected to a low pH wash to remove any surface receptor- bound ligand and incubated with 2 n M '"I-GM-CSF for 1 8 h at 4°C (n=9) . Under baseline conditions, 70% of the cpm bound at this concentration of 1251-GM-CSF are attributable to binding to the low affinity receptor and the remainder to the high affinity GM-CSFR.

two methods: firstly, by measuring the number of cpm specifically bound at two concentrations of I2*I-GM-CSF (100 PM and 2 n M ) and, secondly, by doing full Scatchard analyses across a range of ligand concentrations. Using the data for receptor number and affinity from the Scatchard analysis of equilibrium binding shown above, at 100 PM 12'I-GM-CSF, 80% of cell associated cpm are attributable to binding to the high affinity GM-CSFR and 20% to binding to the low affinity receptor: at 2 nM 'L51-GM-CSF 70% of cpm bound are attributable to binding to the low affinity GM-CSFR and 30% to binding to the high affinity receptor.

(i) Binding at low concentrations of '251-GM-CSF (I 00 p ~ ) . The addition of 5 ng/ml GM-CSF (350 PM) at 37°C caused rapid down-regulation of surface expression of TF- 1 high- affinity GM-CSFR. TF-1 were incubated with 5 ng/ml GM-CSF


- GM-CSFR down-regulation 100 -

80-

E $ 6 0 -

x Q

.- 4 0 -

8 20 -

0 I I I .01 . 1 1 10 100 1 0 0 0

[GM-CSF] pM Fig 4. Concentration-response curves of the effects of GM-CSF on down-regulation of neutrophil GM-CSFR expression and on priming of the neutrophil respiratory burst. Results are shown as percentage of the maximum effects seen with each assay. Neutrophils were incubated with varying concentrations of GM-CSF for 2 h at 3 7OC, washed and then incubated with 1251-GM-CSF for 3 h at 18°C (see Methods) to assess residual surface GM-CSFR (n= 3, mean+range). In parallel experiments, GM-CSF priming of neutrophil H202 production was measured ( n = 3, mean& SE). Also shown is the predicted level of GM-CSFR occupancy by ligand at various ligand concentrations derived from equilibrium binding data of 1251-GM-CSF to neutrophils (1 100f200 R/cell, Kn 50+ 1 5 PM).

2nMGM-CSF

x a

0 1 2 0 3 6 0

Time (minutes) Fig 5. Re-expression of high and low affinity GM-CSF receptors on TF-1 cells after ligand induced down-regulation. TF-1 cells were incubated overnight with GM-CSF (50 ng/ml. 37'C). Cells were then washed extensively and incubated in medium without GM-CSF: aliquots of cells were removed at the indicated times and assessed for bindingto 100p~(n=3,rneaniSE)and2n~(n=2,rnean+range) "jI-GM-CSF to examine re-expression of high and low affinity receptors respectively.

for 4 or 18 h at 3 7°C and the subsequent binding of 100 PM

1251-GM-CSF (following a low pH wash to remove surface- bound ligand) was measured under equilibrium conditions at 4°C. This showed a reduction in cpm specifically bound to 16&4%ofcontrol a t 4 h and 1 2 f 9 % a t 18 h ( n = 3) (Fig 3a). The degree of residual binding a t this low concentration of

1L51-GM-CSF is compatible with binding to the low affinity GM-CSFR only. To confirm this, full Scatchard analyses at these time points were done and showed that there was no detectable binding to the high affinity GM-CSFR at either of these time points and that the cpm bound in the presence of 100 PM 1251-GM-CSF were attributable to binding to the low- affinity receptor (Fig 1).

Experiments with neutrophils, which express high affinity GM-CSFR only, show that residual surface GM-CSFR expression following incubation with 10 ng/ml GM-CSF at 37°C for 2 h was less than 5% of that of control cells (n = 3). The down-regulation of high affinity GM-CSFR is most likely due to the internalization of ligand occupied receptors. Neutrophils were incubated with 100 PM Iz5I-GM-CSF at 4°C for 2 h , washed and shifted to 3 7°C. and measurement of total and low pH resistant cpm (i.e. internalized) carried out. By 9 0 min, 89% of the cpm bound were internal. Internalization was followed by the appearance of non-TCA precipitable radioactivity (detectable a t 120 min) in the supernatant, suggesting release of degraded ligand from the cells (data not shown). A concentration-response curve of the effects of GM- CSF on neutrophil GM-CSFR expression at 37°C (Fig 4) showed that the observed down-regulation of GM-CSFR was significantly greater than that predicted by consideration of static equilibrium binding data, e.g. 100 pg/ml GM-CSF (7 PM) is predicted to occupy - 10% of surface GM-CSFR at equilibrium, but the observed down-regulation of receptor expression at this ligand concentration after 2 h incubation at 3 7°C was 70 f 10% (mean f range).

(ii) Binding at high concentrations of 1z51-GM-CSF (2 nM). Incubation of TF-1 cells with 5 ng/ml GM-CSF a t 3 7°C for 4 or 18 h also caused significant down-regulation of subsequent 2

46 Asim Khwaja et a1 Table I. Neutrophil GM-CSF receptor re- expression after ligand induced down-regulation. Control specific '251-GM-CSF binding at time 0 was 1100+168 cpm/lOh cells. Mean + SE of three separate experiments (time 0 and 18 h are mean of two).

GM-CSF receptor Time expression (h) (% control)

0 3 1 6 + 3 2 4zt2 3 4 f 2

18 2

nM 12TGM-CSF binding to 36 f9% of control at 4 h (n= 9) and 23+5% at 18 h (Fig 3b). This degree of reduction in binding implies that there has been down-regulation of low affinity GM-CSFR as binding to these receptors accounted for approximately 70% of the original cpm bound. This reduction of low affinity receptor expression was confirmed by Scatchard analysis (Fig 1). Increased down-regulation was observed after incubation with 50 ng/ml(3.5 nM) GM-CSF at 37°C for 4 or 24 h with "'I-GM-CSF binding reduced to 1 6 f 2 % of control at 4 hand 9 f 1 % at 24 h (n=4). Down- regulation of iow affinity GM-CSFR is also likely to be due to internalization of ligand occupied receptors: TF-1 cells incubated with 2 nM 1251-GM-CSF at 4°C for 2 h, then washed and shifted to 37"C, showed that 74% of cpm bound had been internalized by 60 min.

Effects of IL-3 and TPA on GM-CSFR expression (i) IL-3. The addition of IL-3 to TF-1 cells (5 ng/ml at 37°C

for 4 h) caused a marked reduction in the binding of 100 PM

1251-GM-CSF to 1 8 f 4 % of control ( n = 5) and also had a significant effect on binding of 2 nM '?'I-GM-CSF which fell to 46 f 5% (n = 8). The binding data at 100 PM '151-GM-CSF are compatible with complete down-regulation of the high affinity GM-CSFR with the residual binding detected attributable to binding to the low affinity receptor. This was confirmed by Scatchard analysis (Fig 1) which also showed a reduction in expression of the low affinity GM-CSFR. As stated above, under equilibrium conditions at 4°C. the cpm bound at 2 n M '"1-GM-CSF are predominantly attributable (70%) to binding to the low affinity receptor, and the reduction seen with IL-3 preincubation suggests that approximately one third of low affinity GM-CSFR are sensitive to IL-3 induced down-regulation at 3 7°C. Increasing the concentration of IL-3 up to 100 ng/ml did not cause further down- regulation of '251-GM-CSF binding. Incubation with IL-3 did not affect 12'I-GM-CSF binding to neutrophils.

(ii) TPA. Addition of TPA (100 ng/ml) at 37°C for 3 h caused significant reductions in subsequent binding of both low (100 PM) and high (2 nM) "'I-GM-CSF concentrations to TF-1 cells (20 f 7% and 44 f 11% of control respectively, n = 3). Incubation with TPA for 30 min at 3 7°C reduced markedly surface GM-CSFR expression on neutrophils (6 f 4% of control, n = 3). No significant effect of TPA on GM- CSFR expression was detected at 4°C in TF-1 cells (92 f 3% of control, n = 3).

GM-CSF priming of the neutropliil respiratory burst A concentration-response curve of GM-CSF priming of the fMLP stimulated respiratory burst showed that half maximal priming occurs at a GM-CSF concentration of 2 PM (Fig 4). This is similar to the concentration of GM-CSF which caused 50% receptor down-regulation.

Re-expression of GM-CSFR following down-regulation Removal of ligand from medium by extensive washing following GM-CSFR down-regulation showed similar rates of re-expression of high and low-affinity receptors on TF-1 cells

S - GM-CSF4h

4 8 0 2 4 4 8 time (hours) time (hours)

Fig 6. Effect on proliferation of TF-1 cells of incubation with GM-CSF (5 ng/ml) for varying lengths of time. TF-1 cells were washed three times to remove GM-CSF from the growth medium and resuspended in medium alone for 18 h. GM-CSF was then added at 5 ng/rnl for 4 h. Cells were then washed extensively and resuspended in mediumfneutralizing anti-GM-CSF antibody (GM-CSF 4 h) or in GM-CSF at 5 ng/ml (continuous GM- CSF). Control cells that were maintained starved but underwent the same washing steps were also resuspended in medium + antibody to control for any non-specific effects. Cells were incubated overnight and then pulsed with %thymidine for 4 h and incorporated radioactivity measured. Viable cell counts were also carried out at 24 and 48 h ( n = 3 , meanrtSD).


(Fig 5). Binding of 100 PM ‘*’I-GM-CSF reached 61+2% (n=3,meanfSE)andof2 nM1”1-GM-CSF 53+12%(n=2, mean frange) of control by 6 h. In contrast, no re-expression of GM-CSFR was detectable on neutrophils up to 18 h following removal of GM-CSF from the medium (Table I).

TF-1 proliferation following exposure to GM-CSF for variable periods To assess the effects of ‘pulsed’ GM-CSF, TF-1 cells were starved of growth factor for 18 h and then GM-CSF was added at either 5 or 50 ng/ml for up to 4 h. Cells were then washed extensively and resuspended in medium + neutralizing anti- GM-CSF antibody (pulsed GM-CSF) or in GM-CSF at 5 ng/ml (continuous GM-CSF). As proliferation was observed in pilot experiments after multiple washes, anti-GM-CSF antibody was added to neutralize any remaining GM-CSF in case that was responsible for the effects seen. Control cells that were maintained starved but underwent the same washing steps were resuspended in medium + antibody. Cells were incubated overnight and then pulsed with 3H-thymidine for 4 h. Viable cell counts were also carried out at this stage. The proliferation assay was repeated at 48 h. Incubation with pulsed GM-CSF at 5 ng/ml for 4 h did not cause significant stimulation of thymidine incorporation at the 24 h time point when compared with control cells: control 2875 f 208 cpm, pulsed GM-CSF 5 ng/mI 4972 f 1344 (P=NS), continuous GM-CSF 5 ng/ml 17249f2982 (P<0.001, n=3) (Figs 6a and 6b). Similarly, a single experiment with pulsed GM-CSF at 50 ng/ml for 4 h did not lead to an increase of thymidine incorporation above control.

DISCUSSION

The data presented in this paper describe the dynamic regulation of GM-CSFR expression and its relationship to cellular responses in neutrophils and in a growth factor- dependent cell line TF-I . Scatchard transformation of equilibrium binding data shows that neutrophils express 110Of 200 high-affinity receptors per cell (KD 50 f 15 PM) and that TF-1 cells have 2000f450 high affinity (KD 15f 5 PM) and 8600* 115010waffinity(KD 1.8f0.3n~)receptorspercell. These figures are consistent with those reported by other workers (Gasson, 1991; Chiba et al, 1990). The GM-CSFR has been shown to consist of two component subunits-the cell surface expression of the CL (ligand binding) chain alone confers low affinity GM-CSF binding whereas the high affinity receptor consists of both CL chain and a non-ligand binding f i chain (Nicola & Metcalf, 1991). The addition of GM-CSF at 37°C to TF-I cells sustained in epo for 24 h caused rapid down-regulation of surface expressed GM-CSFR. Our data show that the binding of lZ5I-GM-CSF to either high or low affinity GM-CSFK on TF-1 cells is followed by rapid internalization. Metcalf et a1 (1 990) have shown that murine FDC-P I cells transfected with the human GM-CSFR CL subunit alone bind GM-CSF with low affinity (& 4-6 nM) and internalize bound ligand slowly, at a rate approximately one-tenth of the human myeloid HL-60 cell line. They postulated that the role of the f i subunit may be to increase the rate of receptor internalization and increase responsiveness to low ligand

concentrations. Our data show that down-regulation of the low a lk i ty GM-CSFR in TF-1 cells following incubation with ligand at 37°C is rapid and concentration dependent with binding of 2 nM IZ51-GM-CSF of 36 f 9% of control following preincubation with 3 50 PM GM-CSF for 4 h and 16 & 2% after 3.5 nM GM-CSF. As occupancy and down-regulation of the high affinity GM-CSFR is complete at the lower concentration of GM-CSF (350 PM) the further down-regulation seen with 3.5 nM GM-CSF suggests that at least a proportion of low affinity GM-CSFRs is rapidly internalized following ligand binding without binding the f i subunit, although the signalling process for internalization could still involve the b chain.

Similarly, rapid down-regulation of high affinity GM-CSFR is seen following the addition of GM-CSF at 3 7°C to neutrophils and we have shown that the observed degree of down- regulation is greater than predicted from consideration of equilibrium binding data. Nicola has suggested that CSF induced receptor internalization at 3 7°C increases the appar- ent affinity of the ligand-receptor interaction by continually removing the reaction product (CSF-receptor complex) from the reaction mixture and has presented supporting data using murine G-CSF (Nicola, 1991). Our data with human GM-CSF support this hypothesis: however, we cannot exclude the possibility that a low level of receptor occupancy may cause down-regulation of unoccupied receptors via intracellular signalling pathways. We have shown that the concentration-effect curve for GM-CSF priming of the neutro- phi1 respiratory burst correlates closely with the observed level of GM-CSFR occupancy/down-regulation at 3 7°C rather than with receptor occupancy predicted by equilibrium binding data. We have also shown that following ligand mediated down-regulation and subsequent removal of GM- CSF from the growth medium, TF-1 cells are able to re- express both low and high affinity GM-CSFR with approximately 50% re-expression within 6 h. In contrast, we have not observed GM-CSFR re-expression on neutrophils over a similar time course. This lack of ability to re-express GM- CSFR in mature cells may serve to limit the effects of this cytokine on neutrophil activation.

The data presented here also show the effects of other molecules on high and low affinity GM-CSFR expression. The b subunit is common to the high affinity GM-CSF, IL-3 and IL- 5 receptors (Nicola & Metcalf, 1991). We were unable to detect any effect of IL-3 on 12TGM-CSF binding to neutrophils and this is consistent with data from other workers showing that IL-3 does not have any effect on neutrophil function (Lopez et al, 1988). In contrast, we have shown that IL-3 will down-regulate both high and low affinity GM-CSFK on TF-1 cells. These cells are reported to have high affinity receptors for IL-3 and the presence of saturating concentrations of IL-3 may deplete a limiting number of p subunits for interaction with the GM-CSFR CL chain (Kuwaki et al, 1989). However, the presence of IL-3 at 37°C also reduced the number of cell surface low affinity GM-CSFR. This down- regulation of low affinity GM-CSF binding was not observed if incubations were carried out at 4”C, suggesting that this is an active process and not due to binding of IL-3 to the low affinity GM-CSFR. The physiological role of this down- regulation is uncertain as IL-3 and GM-CSF are synergistic in

48 Asim Khwaja et a1

their effects on stimulating proliferation of TF-1 cells (Kita- mura et al. 1989). Similar effects have been described in murine bone marrow cells and it has been suggested that this trans-down-modulation may represent activation of these receptors (Walker et al, 1985). We have also shown that treatment with phorbol ester will down-regulate both low and high affinity GM-CSFR. This is consistent with the data of Cannistra et al (1990). The mechanism of this down- regulation is uncertain and may be mediated by receptor internalization or by cleavage of the extracellular binding domain. The latter has been shown for the M-CSFR which is cleaved by a protease that is activated by TPA (Downing et al, 1989). The treatment of TF-1 cells with TPA does not cause the release of a GM-CSF bindable protein into the supernatant (as detected by 1251-GM-CSF competition binding assay: data not shown) but this does not fully preclude GM-CSFR down- regulation by this method.

We have shown that stimulation of TF-1 cells with a 4 h pulse of GM-CSF (up to 50 ng/ml) at concentrations sufficient to occupy all high affinity receptors and the majority of low affinity GM-CSFR did not lead to proliferation which required GM-CSF to be present overnight. This contrasts with data reported by Begley et al (1988) who showed that pulse stimulation for 45 min with high concentrations of GM-CSF or G-CSF could cause limited proliferation of normal human promyelocytes and myelocytes. However, the same authors were unable to detect proliferation when the enriched blast cell fractions from the same bone marrow samples were pulse stimulated by either CSF. TF-1 cells are derived from a patient with erythroleukaemia and are at a stage ofdevelopment that is more akin to these latter blast cells (Kitamura et al, 1989). Therefore, in TF-1 cells, proliferation requires the presence of GM-CSF at a time when no high affinity GM-CSF binding is detectable and the total number of surface GM-CSFR is very low. That the presence of GM-CSF in the medium signals suppression of GM-CSFR surface expression is demonstrated by the rapid re-expression of both high and low affinity receptors following removal of GM-CSF. There are two possible explanations for these observations. Firstly, that the low affinity GM-CSFR may be capable of transducing a proliferative signal. Metcalf described proliferation of murine FDC-P1 cells transfected with the human GM-CSFR LY subunit alone in response to human GM-CSF (Metcalf et al, 1990). This cell line normally responds to murine GM-CSF only. However, Kitamura et a1 (1991a) have shown that the human GM-CSFR LY subunit can interact with the murine GM-CSFR b subunit to produce proliferation in murine CTLL cells that do not respond to human GM-CSF when transfected with the c1 subunit alone. This suggests that the proliferation response to human GM-CSF seen in FDC-Pl cells transfected with the human GM-CSFK LY chain is due to an interaction between the transfected LX chain and endogenous murine f i chain. In addition, Sakamaki et al (1992) have recently shown by co-expressing deletion mutant /3 chains with the LY

subunit of the GM-CSFR that the intracytoplasmic region of the chain between Arg45h and Phe487 is essential for transducing GM-CSF mediated proliferation signals and that the cytoplasmic region of the GM-CSFR LY chain is involved in, but not essential for, signal transduction. These data favour

the second possibility that in TF-1 cells, signalling via the human GM-CSFR involves the chain at a time when there is no detectable surface expression of high affinity GM-CSFR. This may be mediated by transient surface expression of the /j chain with rapid turnover due to new chain synthesis or receptor recycling. This potential mechanism of GM-CSF signalling has significant implications in that it suggests that the absence of high affinity GM-CSFR on cells isolated from normal or malignant tissue does not preclude responsiveness to this growth factor if the cells concerned have been exposed to ligand in vivo, as could occur with autonomous autocrine or paracrine CSF production.

In summary, we have examined the dynamic processes that regulate GM-CSFR expression and have shown that both high and low affinity GM-CSFK are internalized rapidly following ligand binding in responsive cells and that this process may increase the affinity of ligand for receptor. Both classes of GM-CSFR can also be down-regulated in the absence of ligand by incubation with phorbol ester or IL-3 at 3 7°C. Proliferation of TF-1 cells requires the continued presence of GM-CSF at a time when there is no detectable surface high affinity GM-CSF binding. This suggests that signalling can take place via the GM-CSFR at a time when there is no detectable high affinity GM-CSF binding and introduces a further layer of complexity in the analysis of GM- CSF receptor function.

ACKNOWLEDGMENTS

This work was supported in part by the Kay Kendall Leukaemia Research Fund. A.K. is supported by the Well- come Trust.

REFERENCES

Bazan. J.F. (1990) Structural design and molecular evolution of a cytokine receptor superfamily. Proceedings of the National Academy of Sciences o f t k e United States of America, 87, 6934-6938.

Belgley. C.G., Nicola. N.A. & Metcalf, D. (1988) Proliferation of normal human promyelocytes and myelocytes after a single pulse stimulation by purified GM-CSF or G-CSF. Blood. 71. 640-645.

Calvo, J.C., Radicella, J.P. & Charreau. E.H. (1983) Measurement of specific radioactivities in labelled hormones by self-displacement analysis. Biochemical Journal, 212, 259-264.

Cannistra, S.A., Groshek, P., Garlick, R.. Miller, J. & Griffin, J.D. (1990) Regulation of surface expression of the granulocyte- macrophage colony-stimulating factor receptor in normal human myeloid cells. Proceedings of the National Academy of Sciences of the United States of America, 87, 93-97.

Chiba. S., Tojo, A., Kitamura. T., Urabe. A., Miyazono. K. & Takaku, F. (1990) Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colony- stimulating factor. Leukemia. 4, 29-36.

Downing, J.R., Roussel, M.F. & Sherr, C.J. (1989) Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Molecular and Cellzilar Biology. 9,

Gasson, J.C. (199 1) Molecular physiology of granulocyte-macrophage colony-stimulating factor. Blood. 77, 11 31-1 145.

Gearing, D.P., King, J.A.. Gough, N.M. & Nicola. N.A. (1989) Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO Journal. 8, 3667-3676.

2 890-2 8 9 6.

Modulation of GM-CSF Receptor Expression 49 Kuwaki, T.. Kitamura, T., Tojo, A., Matsuki, S., Tamai, Y.. Miyazono.

K. & Takaku, F. ( 7 989) Characterization of human interlenkin-3 receptors on a multi-factor-dependent cell line. Biochemical and Biophysical Research Communications, 161, 16-22.

Lopez, A.F., Dyson. P.G., To. L.B., Elliott, M.]., Milton, S.E., Russell, j.A.. Juttner, C.A.. Yang. Y.. Clark, S.C. & Vadas. M.A. (1988) Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series. Blood, 72, 1797-1804.

Metcalf, D. (1 991) Control of granulocytes and macrophages: molecular, cellular, and clinical aspects. Science, 254, 529-533.

Metcalf, D., Nicola, N.A., Gearing, D.P. & Gough, N.M. (1990) Low- affinity placenta-derived receptors for human granulocyte-macrophage colony-stimulating factor can deliver a proliferative signal to murine hemopoietic cells. Proceedings of the National Academy of Sciences of the United States of America, 87, 4670-4674.

Nicola, N.A. (1991) Receptors for colony-stimulating factors. British Journal of Huematology. 77, 133-138.

Nicola. N.A. & Metcalf, D. (1991) Subunit promiscuity among hemopoietic growth factor receptors. Cell. 67, 1-4.

Sakamaki, K., Miyajima, I., Kitamura. T. & Miyajima, A. (1992) Critical cytoplasmic domains of the common /l subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation. EMBO Journal, 1 1,

Walker. F., Nicola, N.A., Metcalf, D. & Burgess, A.W. (1985) Hierarchical down-modulation of hemopoietic growth factor receptors. Cell, 43, 269-276.

3541-3549.

Hayashida, K . , Kitamura. T., Corman, D.M., Arai, K.-I., Yokota, T. & Miyajima, A. (1990) Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor. Proceedings of the National Academy of Sciences of the United States of America. 87, 9655-9659.

Jawson, M.S., Khwaja, A,, Jones, H.M., Roberts, P.J. & Linch, D.C. (1 990) The effect of rhGM-CSF on the neutrophil respiratory burst in whole blood. British Journal qf Haematology, 75, 181-187.

Khwaja, A.. Roberts, P.J., Jones, H.M., Yong, K., ]aswon, M.S. & Linch, D.C. (1 990) Isoquinolinesulfonamide protein kinase inhibi- tors H7 and HX enhance the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil function and inhibit GM-CSF receptor internalization. Blood, 76, 996-1003.

Kitamura. T.. Hayashida, K., Sakamaki, K.. Yokota, T., Arai, K. & Miyajima. A. (1 991a) Reconstitution of functional receptors for human granulocyte-macrophage colony-stimulating factor (GM- CSF): evidence that the protein encoded by the AICZB cDNA is a subunit of the murine GM-CSF receptor. Proceedings of the National Academy ofSciences ofthe Unitedstates of America, 88, 5082-5086.

Kitamura. T.. Sato, N., Arai, K.4. & Miyajima, A. (1991b) Expression cloning of the human IL-3 receptor cDNA reveals a shared subunit for the human IL-3 and GM-CSF receptors. Cell, 66,1165- 1 174.

Kitamura, T.. Tange, T., Terasawa. T., Chiba. S.. Kuwaki, T., Miyagawa. K.. Piao. Y.-F., Miyazono, K . , Urabe, A. & Takaku, F. (1 989) Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF: IL-3, or erythropoietin. Journal of Cellular Physiology. 140, 323-334.

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Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor