MODIFICATION OF BIOLOGICAL RESPONSES TO INTERLEUKIN-1 BY AGENTS THAT PERTURB SIGNAL TRANSDUCTION PATHWAYS Philippa Rollins, Sara Witham, Keith Ray, Nicola Thompson, Helen Sadler, Nick Smithers, Stephanie Grenfell, Roberto Solari” In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-l). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular CAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-l-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E,. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-l, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate. Copyright o 1991 by W.B. Saunders Company

Interleukin-la (IL-lo) and interleukin-ll3 (IL-lp) are monocyte/macrophage-derived polypeptide hormones that play an important role in a wide range of cellular responses, particularly in inflammation and immunoregulation. Despite only minimal amino acid sequence homology these two cytokines share an almost identical profile of biological activities and bind with equivalent affinity to the same cell surface receptors. An intriguing aspect of the biological responsiveness to interleukin-1 (IL-l) is that cells that express relatively few surface receptors can be shown to respond at concentrations of cytokine at which one would predict low levels of receptor occupancy. Based on studies using CHO cells transfected with the murine IL-1 receptor, it has been estimated that as few as three to seven receptors per cell need be occupied to elicit a biological response.’ Indeed, it has been proposed that certain cells can respond to IL-l even in the absence of surface receptor expression.‘These observations imply that a potent amplification mechanism

Department of Cellular Science, Glaxo Group Research Ltd., Greenford Road, Greenford, Middlesex UB6 OHE, UK. *To whom correspondence should be sent. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0301-0001$05.00/0 KEY WORDS: interleukin-l/IL-l/IL-l 42

receptor/signal transduction

exists for the signal transduction pathway or pathways activated by IL-l. At present there is conflicting evidence linking IL-1 receptor occupancy to second messenger events. IL-1 has been reported to generate a diacylglycerol signal without a concomitant activation of protein kinase C (PKC) or an elevation of intracellular calcium,“’ although recent evidence has shown a potent IL-l-induced activation of PKC in a Th2 cell line.’ The source of the diacylglycerol is still not certain; however, there is evidence that it may be derived from the hydrolysis of phosphatidyl-choline,’ phosphatidyl-inosito14,5-bisphosphate,” phosphatidylethanolamine” or perhaps a novel phosphatidylinositol-glycal moeity.’ IL-l stimulation has also been reported to cause an elevation of intracellular CAMP and an activation of protein kinase A.9,‘2,13Whichever mechanism is involved in signal transduction, downstream events following IL-1 stimulation are beginning to be unraveled. IL-l has been shown to regulate the transcription of several genes, and this transcriptional control may be exerted through DNA binding factors such as NF-KB’~,” and AP1.14,16 In order to further our understanding of the pathways leading from IL-l receptor occupancy to a subsequent biological response, we have examined the effects of agents that are known to modify signal transduction on a range of IL-l-induced events. CYTOKINE,

Vol. 3, No. 1 (January), 1991: pp 42-53

IL-1

RESULTS Interleukin-I

Induction

of Interleukin-2

Secretion

The regulation of IL-2 and IL-2 receptor expression are key features in the activation of T cellsI IL-1 has been implicated in these events, and has been shown to synergize in vitro with agents that deliver signals through the T-cell antigen receptor, although the precise role of this cytokine in vivo is unclear. In murine antigen specific T cell lines such as DlO.G4, IL-l requires an appropriate co-stimulus to elicit its biological response. l8 However, in certain transformed T cell lines such as EL4, IL-l stimulation is sufficient to induce IL-2 secretion.” To investigate the nature of the signal generated by IL-l, we quantified the induction of IL-2 secretion from EL4 cells following stimulation with IL-1 in the presence or absence of agents known to modify second messenger pathways. IL-2 secretion was measured by determination of the proliferation of CTLL cells following their stimulation with EL4conditioned culture supernatant. Typical dose response curves for IL-la and l3 are shown in Fig. 1, demonstrating that both cytokines were equally potent and induced IL-2 secretion with an EC,, of N 250 fM. Equilibrium binding studies with radiolabeled IL-1 revealed that these cells expressed approximately 1,500

i;i 450 I 2 400

II

>: 350 g300

1

.r-i5 250 u : 200 : b 150 u .5 100 i

I I -

50 0 t

=m

0 Cl Figure

= = 1.

IL-l IL-l IL-1

I

I

1

10 Concentration

100 of

IL-l

1000 (fM)

alpha beta induction

of IL-2

secretion

in EL4 ceils.

EL4 cells (1 x lo5 cells per well, in 200 &I) were incubated for 24 h in the presence of increasing concentrations of IL-la (0) or IL-lp (0). Following the incubation, 100 ~1 of the ELA-conditioned supernatant was removed and added toCTLL cells (1 x 10” cells per well, in 100 ~1). The CTLL cells were further incubated for 48 h. and proliferation determined by measurement of ‘H-thymidine incorporation. The two forms of IL-1 were equipotent at induction of IL-2 secretion, with an EC,, of 250 fM.

signal

transduction

/ 43

receptors per cell with a dissociation constant (I(d) of 50 pM (data not shown). We went on to examine the effect of agents that elevate intracellular CAMP on the ability of IL-l to induce IL-2 secretion in EL4 cells (Fig. 2). Forskolin, isobutylmethylxanthine (IBMX), or 8-bromo-CAMP were added to the EL4 cells at the same time as the IL-1 and the cells cultured for 24 h. The supernatants were recovered and their IL-2 content measured in a CTLL assay. All three agents resulted in a dose-dependent inhibition of IL-linduced IL-2 secretion. In order to evaluate further the influence of CAMP and adenylyl cyclase on this biological response to IL-l, we examined the influence of the bacterial toxin cholera toxin. This toxin is known to ADP-ribosylate the alpha subunit of G,, resulting in its permanent activation and coupling to adenylyl cyclase. Consequently, treatment of the cells with cholera toxin would be expected to cause an increased activity of cyclase, and accumulation of intracellular CAMP. Similar to the other agents which elevated intracellular CAMP, cholera toxin caused an inhibition of IL-l-induced IL-2 secretion from EL4 cells even at doses as low as 2 rig/ml (Fig. 3A). Pertussis toxin is known to ADP ribosylate the alpha subunit of the Gi and G, family of GTPbinding proteins resulting in the blockade of receptor/G protein coupling. Treatment of EL4 cells with IL-1 in the presence of pertussis toxin resulted in a total inhibition of IL-2 secretion (Fig. 3B). This effect required pertussis holotoxin since purified binding subunit or A protomer showed no significant inhibition of IL-l-induced IL-2 secretion (data not shown). Activation of protein kinase C, although claimed not to be a direct consequence of IL-1 stimulation, has been shown to potentiate many responses to the cytokine. We consequently examined the effect of the protein kinase C activator dioctanoylglycerol (DOG) on IL-l-induced IL-2 secretion (Fig. 4). At concentrations of DOG from 0.4 to 40 ~J,M, IL-l-induced IL-2 secretion showed a dose-responsive enhancement. This enhancement was most evident when using a combination of a suboptimal IL-1 concentration (0.8 pg/ml) and 40 ~.LM DOG. The resultant IL-2 secretion following such a stimulation was clearly synergistic rather than simply additive. The experimental protocol employed in these studies involved the addition of agents together with IL-1 to EL4 cells and subsequent analysis of the conditioned medium for the secretion of IL-2 by a CTLL bioassay. Clearly there is a possibility that the agents may not be entirely taken up by the EL4 cells and so get carried over into the CTLL assay. To control that the CTLL response was not being influenced by carry over of these agents, we examined their effect on

44 I Rollins et al.

A -ii

CYTOKlNE,

Vol. 3, No. 1 (January 1991: 42-53)

120 r I

.

90 z ----g

0

" 0.8 [ILlal

0 A

- Control = 1pM Forskolin

,

4

0

-

20

[ILlal

100

pg/ml O.lpM X =

0 A

Forskolin 1OpM Forskolin B

:, x -

= Control = 20ng/ml

0 CT

= 2ng/ml X = 200

0.8

4 [ILlal

0 A

0 = Control = lO)lM IBMX

x

20

100

w/ml

1jd.i IBMX = loop IBMX

0 A

CT rig/ml

CT

4oo i

LILhl 0

pg/ml

= Control = 20ng/ml

0 PT

pg/ml

- 2ng/ml X = 200

PT rig/ml

Figure 3. Effect of bacterial toxins on IL-l-induced

PT

IL-2 secretion.

EL4 cells were incubated with increasing concentrations of IL-1 in the presence (open symbols) or absence (0) of (A) cholera toxin or (B) pertussis toxin, and the resultant secretion of IL-2 was measured in a CTLL proliferation assay. Cholera toxin resulted in a reduction of IL-2 secretion, whereas pertussis toxin caused a total inhibition. Each point represents the mean -+ SD of quadruplicate determinations.

&ore 2. Inhibition elevate CAMP.

L .. 0 ’

0.8

4 [ILlpI

0 A

- Control - O.lmM

0 Sbromo

-

20

I 100

pg/ml

O.OlmM &bromo CAMP CAMP X - 1mM B-bromo CAMP

of IL-l-induced

IL-2 secretion by agents that

EL4 cells were incubated with increasing concentrations of IL-1 in the presence (open symbols) or absence (0) of agents that elevated intracellular CAMP levels, and the resultant secretion of IL-2 was measured in a CTLL proliferation assay. Three different reagents were used to determine the effect of elevated CAMP on IL-l induced IL-2 secretion: (A) Forskolin, (B) IBMX, or(C) I-bromo-CAMP. All three reagents were used at three different concentrations. All three compounds resulted in a dose-dependent inhibition of IL-2 secretion. Each point represents the mean + SD of quadruplicate determinations.

IL-1 signal transduction

/ 45

- 250 “0 -4

Interleukin-l-Induced Interleukin-2 Expression in PC 60 Cells

; 200 a E

We wished to examine in a simpler, more defined system, the effects of IL-1 and agents that might alter its signalling pathway. In order to do this we looked at IL-1 signalling in PC60 cells that were stably transfected with the human IL-2 receptor light chain gene under control of the SV40 early promoter and enhancer. These cells are a hybrid between a rat thymoma and a mouse cytotoxic T cell, and the transfectants respond rapidly to IL-l by increasing transcription of the IL-2 receptor reporter gene by up to 30-fold.14,” The TC-II and P motifs in the SV40 enhancer are strongly responsive to IL-l in these cells, and in the case of TC-II this appears to be mediated principally via activation of NF-KB.‘~ The biological response of these cells to IL-l can be quantified by measuring the surface expression of the transfected human p55 IL-2 receptor by FACS analysis. Although the PC60 transfectants show a basal expression of the human IL-2 receptor, this is significantly upregulated by stimulation with IL-l. Figure 5 shows typical dose-response curves for both IL-la and

j7

k 150 ,” F g 100 k :! -- 50 -8g L P Im o-,,, 0 ‘I

=

0.8

4 [ILlal

Control

0

A = 4j~M DOG

0

X

Figure

=

4.

Effect

of PKC

pg/ml

= 0.4yM = 40yM

activation

I 100

20

DOG DOG

on IL-l-induced

IL-2

secretion.

EL4 cells were incubated with increasing concentrations of IL-l in the presence (open symbols) or absence (0) of dioctanoyl glycerol (DOG), and the resultant secretion of IL-2 was measured in a CTLL proliferation assay. Activation of PKC with DOG resulted in a dose-dependent enhancement of IL-l-induced IL-2 secretion. Each point represents the mean 2 SD of quadruplicate determinations.

CTLL proliferation in the presence of added IL-2 (Table 1). At the concentrations used in the above assays, none of the reagents caused a significant inhibition of CTLL proliferation. Table 1. Influence proliferation.

of various agents on ILd-driven

60 (0 l-l rl al u

CTLL

‘0 70 0) u zl ae 60

Percent of control Treatment

Concentration

proliferation

Forskolin

10-l M 1O-6 M 1O-5 M

127.3 122.8 112.7

IBMX

1O-6 M lo-’ M 10m4M

116.7 123.3

lo-’ M 1O-4 M 1O-3 M

116.3 109.3 86.2

DOG

4 x lo-‘M 4 x 10-6M 4 x lo-‘M

118.7 113.9 113.9

Cholera toxin

2 rig/ml 20 @ml 200 rig/ml

97.5 103.3 80.3

Pertussis toxin

2 @ml 20 @ml 200 rig/ml

124.1 126.4 129.1

S-Bromo-CAMP

Receptor

50 L, 0

91.2

,, , “0.01

1 0.1

I 1

[IL11 0

=

Figure 5. Trl17G251

n =

ILla IL1 cells.

induction

I -2 10 100

pM ILlg

of reporter

gene

expression

in PC60

The responsiveness of the SV40 early promoter and enhancer to IL-1 stimulation was examined in PC60 cells stabily transfected with the human IL-2 receptor light chain gene as a reporter under the control of this promoter. Stimulation of the transfectants with IL-la or IL-lp resulted in a dose-dependent upregulation of IL-2 receptor expression on the cell surface as measured by flow cytometry. In these cells, IL-lp was more potent at induction of IL-2 receptor expression (EC,, = 232 fh4) than IL-la (EC,, = 5.6 PM). Each point represents the mean of triplicate determinations.

46 I Rollins et al.

CYTOKINE,

IL-1B. In this system the two cytokines showed differences in EC,,, IL-ll3 (EC,, = 232 fM) being more potent than IL-lo (EC,, = 5.6 PM). IL-2 receptor expression could also be induced by stimulation with 8bromo-CAMP, forskolin, or phorbol ester treatment. However, only phorbol esters gave a level of activation comparable to that of IL-l (Fig. 6). The highest concentration of forskolin used in these studies (10 p,M + 0.1 mM IBMX) resulted in a substantial activation of adenylyl cyclase, and 5 min after stimulation intracellular CAMP levels were increased from a resting level of 1.5 pmols/106 cells to 6 pmols/106 cells, and remained elevated for at least 2 h. However, IL-l stimulation of these cells failed to elevate intracellular CAMP (Fig. 7).

1 OO

Interleukin-I Actions on Human and Murine Fibroblasts Primary cultures of human synovial fibroblasts were established from articular tissues and assessed for secretion of prostaglandin E, (PGE,) in response to IL-l stimulation. A typical dose response to IL-lo and IL-lp (Fig. 8), shows that both cytokines are equipotent at eliciting the PGE, response and have an EC,, of N 2 pM. In order to investigate the signalling pathways implicated in this biological response, we incubated IL-l-stimulated and resting synovial fibroblasts with cholera or pertussis toxins or phorbol ester (Table 2). Stimulation of resting cells with phorbol ester resulted in a small increase in PGE, secretion; however, costimulation of the cells with phorbol ester and 1 pM IL-lo caused a synergistic response that was N 10.5 fold greater than the response to IL-l alone and -7.5-fold greater than to PMA alone. In this system the two bacterial toxins had quite different effects. When administered to either IL-l-stimulated or unstimulated cells, pertussis toxin caused an inhibition of PGE, secretion, whereas cholera toxin augmented the IL-l response. We also measured the IL-l induction of

= =

IBMX. IL1 t

,0-w

,0-l3,0-‘2,0-l!

ILlRtM)

+

accumulation

,

90

60 Minutes

120

IBMX

of cyclic

AMP

in PC60

cells

PC60 transfectants were stimulated with 10 uM Forskolin + 0.1 mM IBMX (W), 50 pM IL-1 + 0.1 mM IBMX (A), or with 0.1 mM IBMX alone (0). Intracellular accumulation of cyclic AMP was determined at timed intervals as described in Materials and Methods. Each point represents a mean of triplicate determinations.

PGE, secretion in Swiss 3T3 fibroblasts. In these cells IL-l causes a dose-dependent increase in PGE, secretion that is unaffected by the presence of 100 r&ml cholera toxin. However, pertussis toxin at a concentration of 10 rig/ml caused an almost total inhibition of the response (Fig. 9). Consequently, in both murine and human fibroblasts the induction of PGE, secretion by IL-l stimulation was inhibitable by pertussis toxin but not by cholera toxin. Swiss 3T3 cells also proliferate in a dose-dependent manner in response to IL-l, a response that has been shown to be a result of the induction of platelet-

L

,070

H=Forskolin IBMX.

Figure 7. Intracellular following stimulation.

I I r

BtW3l

0 A

30

Vol. 3, No. 1 (January 1991: 42-53)

L

loa 105 1Lv 105

107 104 10”

8.Br-cAMP (M)

Forskoltn (Ml +IBMX

1

10 100

PMA lng/mll

Figure 6. Effects of agents on reporter sion in PC60 Trl17G251 cells.

gene expres-

PC60 transfectants were stimulated with increasing concentrations of IL-lB, Sbromo-CAMP, Forskolin (+IBMX), or PMA, and the induction of IL-2 receptor surface expression was measured by flow cytometry as described in Materials and Methods. Each point represents the mean 2 SD.

IL-1 signal transduction / 47

Concentration 0 0

= =

IL-l IL-l

alpha beta

Figure 8. fibroblasts.

IL1

induction

of PGE,

of

IL-l

secretion

from

(PM)

human

synovial

Human synovial fibroblasts were stimulated with increasing concentrations of IL-l. The cell-conditioned supernatants were removed and PGE, quantified by radioimmunoassay as described in Materials and Methods. IL-la (0) and IL-1fJ (Cl) were equipotent at induction of PGE, secretion, with an EC,, of * 2 pM. Each point represents the ,. mean of triplicate determinations. d P’

derived growth factor (PDGF) secretion, which in turn acts in an autocrine fashion as a fibroblast mitogen.” This proliferation of Swiss 3T3 cells in response to IL-l stimulation was unaffected by cholera toxin but was partially inhibited by pertussis toxin (Fig. 10). Since this proliferation induced by IL-l is not a direct effect and depends upon PDGF, we controlled for the effects of the toxins on PDGF-driven proliferation. In such a system neither toxin could be shown to have any effect on PDGF-driven proliferation (data not shown).

Concentration of IL-l @g/ml) Figure blasts.

9.

IL-1

induction

of PGE,

secretion

from

Swiss

3T3

fibro-

Swiss 3T3 fibroblasts were incubated with increasing concentrations of IL-1 and the resultant secretion of PGE, into the supernatant was determined by radioimmunoassay as described in Materials and Methods. PGE, secretion was determined in response to IL-1 alone (0) in response to IL-1 in the presence of 10 @ml of pertussis toxin (0) or in response to IL-1 in the presence of 100 @ml of cholera toxin (A). Due to variability between experiments, the PGE, response has been normalized to “percent of maxima1 response.” In these experiments, the 100% response was approximately 600 fgiml of secreted PGE,. Each point represents the mean + SD of six independent experiments.

DISCUSSION Table 2. Secretion of PGE, by human synovial fibroblasts in response to stimulation with cholera toxin, pertussis toxin, or PMA in the presence or absence of IL-l. Each value represents the mean k SD. PGE, Secretion

Stimulus None Cholera toxin (10 rig/ml) Cholera toxin (100 ngiml) Pertussis toxin (10 rig/ml) Pertussis toxin (100 @ml) PMA (0.1 rig/ml) PMA (1.0 @ml)

-IL-l

2.46 1.30 3.95 1.02 1.12 2.55 9.88

2 t f 2 2 f f

(npil0’ cells) + 1 pM IL-1

1.85 0.09 2.07 0.09 0.10 0.23 4.20

7.13 5.60 15.68 3.10 2.18 5.60 75.15

2 k k k f k k

2.60 0.93 3.06 1.03 0.34 1.60 12.53

IL-1 is known to elicit a wide range of biological responses in many types of target cell, and plays a central role in normal and pathological aspects of immunoregulation and inflammation.22~23 The biological effects of IL-l on target cells are assumed to be a consequence of the cytokine binding to a specific cell surface receptor. This receptor was initially characterized as an 80-kD transmembrane glycoprotein by a series of ligand binding and affinity crosslinking experiments on T cells and fibroblasts.24-29 The receptor was shown to bind both IL-la and IL-1B with high affinity (I& of -50 PM) and in these respects was distinct from a 60-kD form of IL-l receptor that was expressed on the surface of EBV-transformed B cells.30-33Subse-

CYTOKINE,

48 I Rollinsetal

I

I

0

2.5

5.0

, 10

7.5

Concentration of IL-I @g/ml) Figure IL-l.

10.

Proliferation

of Swiss

3T3

fibroblasts

in response

to

Proliferation of Swiss 3T3 fibroblasts in response to increasing concentrations of IL-1 was determined as described in Materials and Methods. Proliferation was measured in response to IL-1 alone (0) in response to IL-1 in the presence of 100 q/ml of cholera toxin (A), or in response to IL-1 in the presence of 10 @ml of pertussis toxin (0). The proliferative response has been normalized to “percent of maximum response,” and represents the means f SD of six independent experiments.

quent cloning and sequencing studies have shown that the IL-l receptor expressed on T cells and fibroblasts is a member of the immunoglobulin gene superfamily,34 and is a distinct gene product from the 60-kD B-cell form of the IL-l receptor.35z36To address the question of receptor function, the cloned 80-kD murine T-cell IL-l receptor was transfected into Chinese hamster ovary (CHO) cells. This transfection confers IL-l responsiveness to these cells and consequently represents strong evidence that this single receptor chain is sufficient to transduce the IL-l signal.’ However, it has been suggested that a simple receptor/ligand interaction of this nature is not sufficient to account for the observed responsiveness of cells to IL-1.2,37”9 Cells that respond to IL-l express relatively few cell surface receptors, and estimates vary from approximately 20,000 to less than 50 per cell. Yet certain cellular responses to IL-l have been demonstrated at concentrations that are several orders of magnitude below the Kd for receptor/ligand interaction,38-40 at concentrations at which receptor occupancy would consequently be limited. Indeed, based on studies of the responsiveness of CHO cells transfected with the IL-l receptor, it has been estimated that as few as three to seven receptors per cell need be occupied to induce a response.’ The ability of IL-l to elicit biological responses at

Vol. 3, No. 1 (January 1991: 42-53)

such low levels of receptor occupancy raises questions as to the nature of the signal transduction mechanism that is coupled to the IL-l receptor. In T cell activation, it has been suggested that IL-l appears to act as a co-stimulus with signals generated through the T-cell antigen receptor/CD3 complex in the induction of IL-2 and IL-2 receptor light chain expression.18,19,41-44 Activation of the T-cell antigen receptor/CD3 complex results in phosphatidyl-4,5,-inositol-bisphosphate (PIP,) hydrolysis, with consequent elevation of intracellular calcium and diacylglycerol levels, and activation of protein kinase C (PKC).45 Such a stimulus is sufficient to induce cytokine production, but the IL-l co-stimulus is required to induce cytokine receptor expression and so to activate fully the T cell.‘s,41,UParadoxically, the IL-l co-stimulus can be replaced by phorbol esters,e’47 suggesting that IL-l stimulation perhaps results in the activation of a PKC distinct from the PKC activated by T-cell antigen receptor/CD3 triggering. However, IL-l signal transduction cannot be explained simply by activation of a PKC, for although phorbol esters are able to substitute for IL-l in certain situations, there are some distinct differences in their mode of action.9*16,44 Identification of the early biochemical events following IL-l receptor occupancy remains unclear and the subject of a degree of controversy. There is evidence that IL-l induces the generation of diacylglycerol (DG), potentially from the hydrolysis of phosphatidylcholine,’ although there is a suggestion that the DG generated in response to IL-1 contains myristic acid and may consequently derive from the hydrolysis of a phosphatidylinositol glycan.8 The total absence of a calcium signal would appear to rule out hydrolysis of PIP, as the source of DG. Moreover, although IL-l generates a DG signal, it has not been possible to demonstrate the activation of PKC in most cases2-9 although there is recent evidence for such an activation.9 Further support for IL-l triggering the PKC pathway comes from studies on the immunosuppressive activity of the retroviral p15E protein, which appears to inhibit both PKC and IL-1 signal transduction.48 Receptor occupancy has been demonstrated to induce the phosphorylation of a range of substrates on serine and/or threonine residues. The EGF receptor becomes rapidly phosphorylated on serine residues, by an apparently PKC-independent mechanism, resulting in a decreased affinity for its ligand.49 IL-l also induces phosphorylation of a 2%kD heat shock proteinjo and a 65kD cytosolic protein5’ and the IL-l receptor itself becomes phosphorylated following ligand binding.52 Further evidence linking IL-l receptor occupancy to increased protein kinase activity are the findings that the transcription factors AP114,16and NFKB’~.” become activated. Although both of these factors are consid-

IL-1 signal transduction / 49

ered to be activated through the PKC pathway, there is evidence that NFKB can be activated by protein kinase * (PI(A),13,53-55 and that IL-l may indeed be acting through adenylyl cyclase to elevate intracellular ,AMp.9,‘2.‘3 In the studies reported here, we have investigated biological responsiveness of a range of cells to IL-1 stimulation, and the modification of those responses by agents that are known to perturb signal transduction pathways. The murine thymoma EL4 has been extensively used as a tool to study the biology of IL-1 since these cells can be induced to secrete IL-2 in response to IL-l stimulation. We have confirmed the finding of other groups that activators of PKC such as phorbol esters or synthetic diacylglycerols greatly enhance this response. At suboptimal concentrations, the combination of IL-l and DOG clearly resulted in a synergistic induction of IL-2 secretion, as has been shown previously for IL-1 and phorbol esters.19 On the other hand, agents that cause an elevation of intracellular CAMP inhibited the ability of IL-l to elicit IL-2 secretion. These findings are in agreement with previous studies that have shown the inhibitory effect of elevated intracellular CAMP on IL-2 production by T cells.56*57 Consequently in T cells it is unlikely that IL-l is acting to elevate CAMP levels. Furthermore, we have been consistently unable to measure IL-l-induced elevation of intracellular CAMP in these or in PC60 cells. The bacterial toxins, pertussis toxin and cholera toxin, both inhibited IL-l-induced IL-2 secretion; however, their respective actions may be at different sites in the signalling pathway leading to the induction of IL-2 secretion. We deduce from our observations that pertussis toxin is acting at the level of the IL-l receptor coupling to its effector mechanism because its inhibitory action is seen on all biological responses to IL-l stimulation, not only IL-l induction of IL-2 secretion. Cholera toxin, on the other hand, may be exerting its inhibitory action on IL-2 production through the direct activation of adenylyl cyclase and elevation of CAMP levels because cholera toxin is capable of blocking this response to IL-l but not others that appear to be CAMP independent. Since these studies depended upon the proliferation of CTLL cells in response to EL4-conditioned medium to quantify IL-2 secretion, we controlled the action of the various agents employed directly on IL-2-driven CTLL proliferation. Even assuming a 100% carry over of the agents from the EL4 supernatant to the CTLL cells, none of the agents significantly affected the proliferative response. It is well established that in fibroblasts IL-1 is a potent activator of phospholipase A, (PLA,) and the subsequent secretion of PGE2.58z59 We have examined this response in human synovial fibroblasts and Swiss 3T3 fibroblasts and the influence of pharmacological

agents upon IL-l activation. In the human synovial fibroblast assays, IL-la and IL-1B were equipotent in induction of PGE, secretion with an EC,, of 2.1 pM and 1.6 pM, respectively. The EC,, for this IL-l response is close to the I& for receptor/ligand interaction and, consequently, is quite different from the potency of IL-l on T cells. In the absence of IL-l stimulation there is a constitutive basal level of PGE, secretion, which is unaffected by either cholera or pertussis toxins. Phorbol ester treatment at a concentration of 1 rig/ml did, however, result in a significant increase in secretion. When tested in the presence of 1 pM IL-l, cholera toxin enhanced the IL-l response whereas pertussis toxin was inhibitory, and the phorbol ester response in the presence of IL-1 showed marked synergy. The same pattern of toxin sensitivity was seen in the PGE, response of Swiss 3T3 cells. The IL-l response was unaffected by treatment with cholera toxin, whereas pertussis toxin almost totally abolished PGE, secretion. The IL-1 induction of PGE, secretion from fibroblasts would appear to result from the stimulation of both PLA, and cyclooxygenase synthesis,58.60and the latter has been claimed to be dependent upon PKC activation by IL-1.6o IL-l also induces proliferation in Swiss 3T3 cells, a response that has been shown to be an indirect effect mediated through IL-l induction of PDGF secretion.” These cells proliferate in a dose-dependent fashion in response to IL-l stimulation, and this proliferation was partially inhibited by pertussis toxin, but was unaffected by cholera toxin. Control experiments showed that pertussis toxin was not acting to block PDGF signalling events. Consequently in both murine and human fibroblasts biological responses to IL-l stimulation were blocked by pertussis toxin, but not by cholera toxin. The aim of many recent studies of IL-1 signal transduction has been to understand how the biochemical events initiated by receptor/ligand interaction are converted further downstream into the activation of the appropriate target genes. The expression of several genes have been shown to be controlled by IL-l, and implicated in these events has been the activation of the DNA binding proteins NFKB’~-‘~ and AP1.‘4,‘6 Although both IL-l and phorbol esters have been shown to activate these transcription factors, there are subtle differences between the pathways involved.16 Moreover, it has been suggested that IL-1 may activate NFKB via a novel mechanism involving the elevation of intracellular CAMP and PKA.13 In order to investigate these events we have examined the IL-l responsiveness of the SV40 enhancer transfected into an immature rat/mouse T cell hybridoma.14s20 In this cell line, IL-l and tumor necrosis factor (TNF) have been shown to increase the expression of a human IL-2 receptor light chain reporter gene, and this effect was mediated

CYTOKINE, Vol. 3, No. 1 (January 1991: 42-53)

50 I Rollins et al.

principally through the activation of NFKB. We first performed dose response curves for IL-lo and IL-lB and demonstrated that IL-la was less potent (EC, = 5.6 PM) than IL-@ (EC,, = 232 fM). This may reflect the specificity of the rat IL-l receptor on these cells. The potency of IL-@ in these cells is surprising considering that these cells express only about 25 to 50 receptors per cell (data not shown). In order to examine the signalling pathways involved in this activation event we demonstrated that both IL-l and phorbol ester were effective in induction of reporter gene expression. However, agents that gave substantial and sustained increases in intracellular CAMP concentrations gave only marginal responses, suggesting that IL-l was not signalling through such a pathway in these cells. Our inability to measure detectable increases in intracellular CAMP levels following stimulation with IL-l confirmed these findings. Consequently, in these PC60 cells it seems probable that IL-l is triggering the SV40 enhancer by activation of NFKB via a pathway common to PKC rather than to PKA. In conclusion, our data is consistent with the T cell/fibroblast IL-l receptor being coupled via a pertussis toxin-sensitive GTP-binding protein to a signal transduction mechanism generating second messengers that result in the activation of a kinase. Whether IL-1 activates PKC, PKA, or another kinase remains unclear, and there is conflicting evidence on this matter. However, based on our studies, IL-1 would appear to activate effector mechanisms within the cell that are common to the PKC pathway rather than the PKA pathway. There is sufficient evidence that “crosstalk” between the IL-l receptor and other signalling pathways exists, and that the actions of IL-l should not simply be interpreted as the activation of a single kinase. The marked synergy observed when cells are stimulated with suboptimal concentrations of IL-l and agents that activate PKC suggests that there are additional components to IL-l signalling that remain to be resolved. Nevertheless, we find no evidence that IL-l signalling in T cells can be accounted for by activation of PKA. Indeed, elevation of intracellular CAMP in T cells appeared to inhibit IL-l induction of IL-2 secretion. Further studies are underway to dissect these signalling events.

MATERIALS AND METHODS Reagents Human recombinant IL-la, IL-l& and IL-2 were obtained from the Glaxo Institute of Molecular Biology, Geneva, Switzerland. The anti-human IL-2 receptor antibody (CD25) and the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin were obtained from Becton Dickinson. [‘HI-PGE,, scintillation proximity assay beads, and [met/zyZ-3H]thymidine were from Amersham Interna-

tional (Amersham, Buckinghamshire). The following reagents were obtained from Sigma (Poole, Dorset): forskolin, isobutylmethyl xanthine (IBMX), 8-bromoadenosin 3’:5’cyclic monophosphate (8-bromo-CAMP), phorbol-12myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), cholera toxin, PGE,, and anti-PGE, antiserum. Pertussistoxin was obtained from Porton Down, and pertussistoxin subunits were obtained from List Biological Laboratories Inc., California. Cyclic AMP radioimmunoassay kits were obtained from NEN DuPont (Stevenage, Hertfordshire).

Cell Lines Wild type PC60 cells and the Tr17G251 transfectants were a kind gift from Dr. Markus Nabholz of the Swiss Institute for Experimental Cancer Research. EL4.6.l.dlO cells were a kind gift from Dr. Robson MacDonald of the Ludwig Institute, Epalinges, Switzerland. The cytotoxic T cell line (CTLL) was obtained from the American Tissue Culture Collection, and were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 x 10m5M 2-mercaptoethanol(2-ME), 2 mM glutamine, and 2 U/ml of rat IL2. Cells were subcultured three times per week. Human synovial fibroblasts were obtained from explants or from digests with bacterial collagenase of material obtained during hip joint replacement surgery on osteoarthritic patients, as previously described,6’ and were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FCS. Swiss3T3 fibroblasts were obtained from Flow Laboratories (Irvine, UK) and were cultured and passaged as previously described.62 The PC60 cells were maintained in DMEM supplemented with 5% heat-inactivated FCS, non-essential amino acids, and 20 ~J,M2-ME (Gibco). EL4.6.ldlO cells were maintained in RPMI-1640 medium supplemented with 10% FCS (Flow), 20 JLM 2-ME, and 2mM glutamine.

Proliferation Assays IL-l induction of IL-2 secretion from EL4 cells was measured by incubating EL4 cells overnight in the presence of IL-l and measuring the release of IL-2 into the culture medium using a CTLL proliferation assay.The EL4 incubation was performed in a 96-well plate at a concentration of 1 X lo5 cells per well in a total volume of 200 i.~l.IL-2 was secreted into the cell supernatant and this was quantified by induction of CTLL proliferation. The CTLL cells were prepared for the assayby being removed from maintenance culture at least 48 h after receiving IL-2, washed three times in RPMI-1640 medium supplemented with 5% FCS and 2-ME, and resuspended at lo5 cells/ml. 100 l.~laliquots of the cell suspension were added to each well of a 96-well microtiter plate. Samples of IL-2 containing EL4 cell conditioned medium were diluted in assaymedium and added in quadruplicate to the microtiter plate in 100 ul aliquots. Recombinant human IL-2 was used as a standard preparation at concentrations between 1 and 1,000 pg/ml. The plates were incubated for 24 h at 37°C and 10 l.~l (37 KBq) of ‘Hthymidine was added to each well prior to a further overnight incubation at 37°C. Cells were harvested onto glassfiber filter

IL-1 signal transduction

mats with the use of a Scatron Cell Harvester and the mats were dried at 80°C. Ten milliliters of scintillant were added to each filter mat and incorporation of 3H-thymidine determined using an LKB 1205 B-Plate spectrometer. Proliferation of Swiss3T3 fibroblasts in response to IL-l or PDGF was determined as follows: 3T3 cells were resuspended in DMEM supplemented with 10% donor calf serum, plated out at a density of 2.5 x 10“ cells/well in a 48-well plate and maintained for 6 days in culture to reach confluence. The cells were washed three times in phosphate-buffered saline (PBS), and 500 l.r,lof assay medium (DMEM/Waymouths medium [l:l, v/v], supplemented with 0.5% BSA) containing IL-l or PDGF. After an 18 h incubation, 20 ~1of a thymidine cocktail (DMEM supplemented with 25 t&i/ml ‘H-thymidine, 10 FM unlabeled thymidine) was added to each well. After 6 h, the medium was removed and discarded. The cells were washed twice with ice cold 5% trichloroacetic acid, and stored at 4°C for 18 h in the presence of 5% trichloroacetic acid. Following two washes with distilled water, the bound counts were solubilized by the addition of 250 t~J0.2M NaOH to each well, and incubation at 37°C for 1 h. The alkali was neutralized by the addition of 25 )~l2M acetic acid, and the contents of each well transferred to scintillation vials containing 3 ml of scintillant. The 3H incorporated into the cells was determined using an LKB 1219 Rackbeta liquid scintillation spectrometer.

Prostaglandin

E, Assays

Synovial cells were plated out at a density of 10” cells/well in a 96-well plate and maintained for 3 to 5 days in culture to reach confluence. The cells were washed with fresh medium, then stimulated with IL-l or compounds for a further 24 h. Following this incubation, 150 ~1 of the culture medium was removed from each well and stored at -20°C until assayed. The level of PGE, released into the culture supernatant was assayedby a radioimmunoassay using [jH]PGE, as a standard. Briefly, samples of the cell-conditioned medium were diluted in assay buffer (O.lM sodium phosphate buffer, pH 7.2, 0.15 M NaCl, 0.1% bovine serum albumin, and 0.1% NaN,) and to 100 t.i,lof each sample was added 500 l~,lof diluted anti-PGE, antiserum and 100 l.r.1of diluted [jH]-PGE,. The assaymixture was incubated for 18 h at 4°C and the reaction terminated by the addition of 200 l~,l of dextran/charcoal (50 ml of assay buffer, 50 mg Dextran, 500 mg charcoal). Each tube was vortexed and centrifuged for 20 min at 1,3OOg,,.Supernatants were removed and mixed with scintillant for determination of 3H in an LKB 1219 Rackbeta scintillation spectrometer. Each determination was performed in triplicate or quadruplicate, and the quantity of PGE, present in the cell-conditioned medium was calculated by comparison with a PGE, standard calibration curve. Scintillation proximity assaywas used as an alternative to the Dextran/charcoal precipitation procedure. 100 ul of the assaymixture was placed in each well of a 96-well T-tray, and incubated with 100 ul of Protein A-coated scintillation proximity assay (SPA) spheres (1 vial reconstituted in 50 ml of assaybuffer), 100 ul 3H-PGE, (16 nCi/well), and 100 ~1 anti-PGE, antiserum, on a shaker, for 4 h at room temperature. The quantity of [‘HI-PGE, bound to the SPA spheres for each sample was determined by a scintillation spectrome-

/ 51

ter, and from this was determined the PGE, content of the cell-conditioned medium by comparison with a standard calibration curve.

Flow Cytometry Analysis Cells (0.5 x lo6 to 1 x lo6 in 2 ml) were incubated for 18 h with either IL-l or an appropriate reagent. Induction of IL-2 receptor expression was subsequently measured with a Beckton-Dickinson FACS analyzer as described previously.63 Live cells were gated by forward and right angle light scatter, with 5,000 events collected by the computer. In order to express the results from the PC60 cells, where there was significant expression of IL-2 receptor under non-stimulated conditions, the cells were gated such that approximately 50% of the untreated cells had a fluorescence greater than the gated value.

Cyclic AMP Assays PC60 cells were aliquoted in culture medium into 24-well plates at a density of 2 x lo6 cells per well in a volume of 1.5 ml. At the zero time point IL-l@, forskolin, and IBMX were added to each well as appropriate. Cells were incubated at 37°C for specific times and then harvested by centrifugation at 17,OOOg,,for 1 min. Cell pellets were rapidly resuspended in 1 ml of ice-cold 0.2 M HCl and incubated on ice for 1 h. Samples were recentrifuged as above and the supernatants removed and stored at -20°C. Prior to assayfor CAMP levels, samples were neutralized with 200 ~1of 0.97 M NaOH and 0.5 M sodium acetate, pH 6.2. Samples were subsequently assayedfor CAMP by radioimmunoassay according to the manufacturers instructions.

Acknowledgment The authors would like to thank Dr. Markus Nabholz for his generous donation of the PC60 cell line and for his valuable advice. REFERENCES 1. Curtis B, Gallis B, Gverell R, McMahan C, DeRoos P, Ireland R, Eisenman J, Dower S, Sims J (1989): T-cell interleukin 1 receptor cDNA expressed in Chinese hamster ovary cells regulates functional responses to interleukin 1. Proc Nat1 Acad Sci USA 86:3045-3049. 2. Rosoff P, Savage N, Dinarello C (1988): Interleukin-1 stimulates diacylglycerol production in T lymphocytes by a novel mechanism. Cell 54:73-81. 3. Avissar S, Stenzel K, Novogrodsky A (1985): Selective effects of TPA and IL-1 on protein phosphorylation in murine thymocytes. Cell Immunol96:462-471. 4. Avissar S, Shanitzki B, Stenzel K, Novogrodsky A (1986): Early effects of TPA on protein kinase activity in murine thymocytes. Exp Cell Res 165:353-361. 5. Abraham R, Ho S, Barna T, McKean D (1987): Transmembrane signaling during interleukin l-dependent T cell activation. J Biol Chem 262:2719-2728. 6. Ho S, Abraham R, Nilson A, Handwerger B, McKean D (1987): Interleukin l-mediated activation of interleukin 4 (IL4) producing T lymphocytes. Proliferation by W&dependent and IL 4-independent mechanisms. J Immunol 139:1532-1540.

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