ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 295, No. 1, May 15, pp. 42-48, 1992
Stimulation of Macrophage Synthesis by Interleukin-I
Colony-Stimulating
Jean C.-Y. Ku, Mu-Ya Liu, and Ming-chi
Wul
Factor
Department of Biochemistry and Molecular Biolozy, Texas College of Osteopathic Medicine/ Urkersity of North Texas-Fort Worth, Texas 76107
Received November
12, 1991, and in revised form January
17, 1992
Interleukin-1 (IL-l), which plays an important role in the inflammatory response, was found to induce colonystimulating factor-l (CSF-1) expression in the MIA PaCa2 cells. IL- l-induced CSF- 1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-l-induced CSF1 production was inhibited by cholera toxin and this inhibition was reversed by an a&nine analog, pmethoxybenzylaminodecamethylene guanidine sulfate. DibutyrylCAMP as well as other CAMP elevating agents such as theophylline and forskolin also suppressed IL-l-induced CSF-1 production, suggesting that CAMP concentrations inversely regulate the biosynthesis of CSF- 1. Measurement of CAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the CAMP level. IL-linduced CSF- 1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12-0-tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL- 1 -induced CSF- 1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF- 1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression. 0 1992 Apress. Inc.
Although the production of CSFs2 by recombinant DNA techniques has been accomplished and CSFs in general 1 To whom correspondence should be addressed. ’ Abbreviations used: CSF, colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-l, interleukin-1; TPA, 12-0-tetradecanoylphorbol-13-acetate; DME, Dulbecco’s modified Eagle’s; PBS, phosphate-buffered saline; SSC, standard saline citrate; SDS, sodium dodecyl sulfate; MBAMG, p-methoxybenzylaminodecamethylene guanidine sulfate; RIA, radioimmunoassay; TCA, trichloroacetic acid, DMSO, dimethyl sulfoxide; PKC, protein kinase C; TNF, 2,4,7-trinitrofluorenone; PTX, pertussis toxin; BZ, benzamide. 42
have been reviewed recently (1, 2), the molecular mechanisms involved in the regulation of their synthesis have not been investigated systematically. A variety of cell types such as macrophages, T-lymphocytes, endothelial cells, and fibroblasts can produce CSFs, particularly under cytokine stimulation (3-5). Bacterial endotoxin has long been used as the stimulator for CSF production. Other mitogens have also been shown to stimulate CSF production. IL-l is a member of the monokine family produced primarily by monocytes. It is a key mediator of the host response to infections, inflammations, and immunologic challenges. Monocytes, T-lymphocytes, B-lymphocytes, hepatocytes, neutrophils, fibroblasts, and endothelial cells can be stimulated by IL-l to generate many features of the inflammatory reaction (3-5). The effects of acute and chronic inflammation on the hematopoietic system include release of neutrophils from the vascular marginal pool; premature release of neutrophils from the marrow into the circulation; enhanced neutrophil chemotaxis to, and activation at, sites of inflammation; and a sustained increase in neutrophil and monocyte production. Many of these responses are evoked by the various CSFs in vitro. Zucali et al. (3) have shown that IL-l-stimulated fibroblasts produce GM-CSF and prostaglandin E2. Segal et al. (4) and Zsebo et al. (5) have reported that ILl-stimulated endothelial cells release multilineage human CSF activity in addition to GM-CSF as previously reported. Ralph et al. (6) have also reported that TPA stimulated CSF-1 production from MIA PaCa-2 cells and that the increase of CSF-1 production is correlated with mRNA expression. However, the mechanism involved in the regulation of CSF production stimulated by monokines and other stimuli is not very well understood. Furthermore, whether the regulation is at the transcriptional or translational level remains to be studied. The human pancreatic carcinoma cell line MIA PaCa2 established by Yunis et al. (7) has been a rich source for CSF-1 (8, 9). This cell line has been used to clone CSF-1 cDNA (10) and is used in this experiment to study 0003-9S61/92
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
INDUCTION
OF MACROPHAGE
COLONY-STIMULATING
the mechanism involved in the stimulation of CSF-1 production by IL-l. Data presented in this article indicate that CSF-1 synthesis is inversely regulated by CAMP levels. Although the role of the IL-l-induced signal transduction pathway in enhancing CSF-1 expression is still not clear, the involvement of protooncogene c-fos and cjun expression is suggested. MATERIALS
AND
METHODS
Cell culture. The MIA PaCa-2 cells were cultured in DME (Dulbecco’s modified Eagle’s) medium and supplemented with 5% fetal calf serum, 3% newborn calf serum, penicillin (100 pg/ml), and streptomycin (100 ag/ml) as described previously (8). The confluent cultures were changed to serum-free DME medium. Different concentrations of lymphokine and other modulators were added to the culture. The incubation was then continued for different periods of time, depending on the culture conditions of each experiment. The conditioned medium thus prepared was assayed for CSF-1 activity as described below. Mouse marrow assay for CSF. The conditioned media prepared under different stimuli were assayed for CSF-1 activity using the mouse bone marrow method (9). Mouse bone nucleated cells (10s) obtained from C57 BL/6 mice (Jackson Laboratories, Bar Harbor) were added to a final volume of 1.2 ml soft-agar medium with 50 al of conditioned medium. The agar cultures were then incubated at 37°C under 6% CO, for 5 days. Aggregates of 50 or more cells were counted as colonies using a dissecting microscope. RNA isolation and Northern blot analysis. Confluent MIA PaCa-2 cells were cultured with or without IL-1 for various times. The cells were washed twice with phosphate-buffered saline (PBS). Total RNA (20 pg) extracted by the guanidium/cesium chloride method (11) was denatured and size-fractionated by 1% formaldehyde-agarose gel electrophoresis and transferred to a nylon membrane by capillary blotting using 20X SSC. For the equality of RNA loading, parallel RNA samples were stained with ethidium bromide to visualize 28s and 18s ribosomal RNA bonds. The membrane was irradiated with uv light for 5 min. Prehybridization was performed in 5X SSC, 15X Denhardt’s, 0.1% SDS, and denatured salmon sperm DNA at 65°C for 18 h. The blot was hybridized with y-32P-labeled 40-mer synthetic probe (CSF-1, c-fos, and c-jun) purchased from Oncogene Science, Inc. Hybridization was performed in the same buffer for another 18 h at 65°C. Membranes, washed with 1X SSC and 0.1% SDS at room temperature, 42°C and 65°C for half an hour each time, were subjected to autoradiography at -70°C by exposing to Kodak XAR5 film with an intensifying screen. Recombinant IL-I. Recombinant IL-lo (rHu. IL-la [117-2711 Ro245008) (gift from Hoffmann-LaRoche, Inc., Nutley, NJ) was added to the cell cultures in the concentration range of 0.7 to 700 rig/ml. The activity is 2 X 10s U/ml by DlO cell thymidine uptake assay. Effectof IL-l on CSF-1 production. After confluence, growth medium was removed from MIA PaCa-2 cells. The cells were washed with PBS and DME serum-free medium was added. IL-l at concentrations of 0, 0.7, 7.0, 70.0, and 700.0 rig/ml was added to the culture plates and incubated for an additional 72 h. The media were harvested, desalted by PD.10 (Pharmacia), sterilized by filtration, and assayed for CSF-1 activity in triplicate plates. Time course of IL-l-induced CSF-1 production. Confluent MIA PaCa2 cells in DME serum-free media were cultured with IL-l (7 rig/ml) for different periods of time (0, 4, 8, 16, and 20 h) or without IL-l for 20 h as control. The media were harvested, desalted by PD-10, sterilized by filtration, and assayed for CSF-1 activity. Effect of pertussis toxin and cholera toxin on IL-l-induced CSF-1 production. Cultured MIA PaCa-2 cells in serum-free DME medium were incubated with IL-1 (7 rig/ml), pertussis toxin (200 rig/ml, List Biological Laboratories, CA), and benzamide (2 mM) separately or in combination for 72 h. The conditioned media were harvested and assayed for CSF-
FACTOR
BY INTERLEUKIN-1
43
1 activity in triplicate plates. For the cholera toxin experiment, IL-ltreated MIA PaCa-2 cells were incubated with cholera toxin (10 to 1000 pg/ml) and 5 FM MBAMG, as in pertussis toxin experiments. Effectof dibuty$cAMP on IL-l -induced CSF-1 production. Cultured MIA PaCa-2 cells in serum-free DME medium were incubated with or without IL-l (7 rig/ml) and dibutyryl-CAMP (10-r M) for 48 h. The stock solution of dibutyryl-CAMP (10e3 M) was prepared in absolute ethanol. The conditioned media were assayed for CSF-1 activity as described above. Radioimmunoassay of CAMP. The level of CAMP from the CSF-1 stimulation or inhibition study is measured by radioimmunoassay (RIA) as described by Hopkins and Gorman (12). Briefly, cells (3 X 10s) in tissue culture plates are treated with or without 1 mM theophylline at 37’C for 10 min prior to adding effecters. At the end of incubation with the addition of effecters (30 min), 0.5 ml of 10% TCA is added and aqueous samples are extracted three times with 10 vol of diethyl ether. The aqueous phase is evaporated and the residue dissolved into 50 mM sodium acetate buffer, pH 6.2. The RIA of CAMP is carried out as suggested by the manufacturer (NEN Research Products, NEK-033). Effect of theophylline and forsholin on CSF-1 production. Theophylline and forskolin, which can increase the cellular CAMP level by inhibiting phosphodiesterase and stimulating adenylate cyclase, respectively, are used to further study the role of CAMP in regulating CSF-1 synthesis. The stocks of theophylline and forskolin are prepared in 100% DMSO and absolute ethanol, respectively. Confluent MIA PaCa2 cells in serum-free DME media were incubated with 0, 0.01, 0.1, and 0.5 mM of theophylline (Sigma) or 1.65, 3.3, and 6.6 pM of forskolin (Sigma) to a final volume of 2.5 ml. After 18 h incubation, the medium was harvested and assayed for CSF-1 activity. Effect of H-7 and HA1004 on IL-l-induced CSF-1 production. Two protein kinase inhibitors were used in this study to differentiate whether the IL-l-induced CSF-1 production involved the CAMP-dependent protein kinase or protein kinase C process as the signal transduction mechanism. To petri dishes with confluent cultured MIA PaCa-2 cells was added serum-free DME medium, which was then incubated with 25 pM H-7 or HA1004 (Seikagaku America, Inc., St. Petersburg, FL) for 2 h before IL-1 (7 rig/ml) or TPA (10 rig/ml) was added. Incubation was continued for 18 h and the media were harvested and assayed for CSF1 activity. The stock solutions of H-7 and HA1004 were 10 mM in distilled water. Data analysis. Data are presented as the mean + SD with triplicate determination or given assay parameters from separate dishes within each experiment and normalized to concurrent control cultures.
RESULTS
IL-1 -Induced CSF-1 Production As shown in Fig. 1, cultured nonstimulated MIA PaCa2 cells (control) produced low levels of CSF as measured by the mouse marrow soft agar assay. By dose-response analysis, the addition of increasing concentrations of IL1 (0.7 to 700 rig/ml) enhanced the production of CSF-1 in the medium by about threefold. The concentration of IL-l that resulted in maximum stimulation was around 7.0 rig/ml. Therefore, the IL-l concentration of 7.0 ng/ ml was used for the rest of the study. The control experiment was carried out to check if IL-l has any colonystimulating activity alone or synergistic effect on colony formation with CSFs. The result showed that IL-l had no direct effect on colony formation nor any synergistic effect with CSF at these concentrations (data not shown). As shown in Fig. 2a, IL-l-induced CSF-1 activity was first detected between 0 and 4 h, and continued to accu-
44
KU,
LIU,
AND
WU
200
Y
b
IL-1
-
+
+
hr
-
.5
3
1
2
3
150
z 60
100
3 s
50
0
0
0.7
7
CONC.
OF IL-I
70
700
0
(rig/ml)
FIG. 1. Stimulation of CSF-1 production by IL-l. MIA PaCa-2 cells were cultured until confluence. The growth medium was removed, the cells were washed with PBS, and serum-free medium was added. IL-1 at different concentrations was then added and the incubation was continued for 72 h. The media were harvested, desalted, sterilized by filtration, and assayed for CSF-1 activity in triplicate plates.
mulate in the medium over 20 h. A fourfold stimulation over the control was observed at 20 h. By Northern blot analysis, IL-l also increased accumulation of CSF-1 mRNA levels after 3 h treatment (Fig. 2b, lane 3), compared with the control which was without IL-l treatment (Fig. 2b, Lane 1). The increase of CSF-1 activity in the medium is consistent with the increase of mRNA as induced by IL-l, suggesting the stimulation of CSF-1 expression at the transcriptional level.
Inhibition of IL-l-Induced Pertussis Toxin
CSF-1 Production
by
In order to examine whether G-proteins are involved in the IL-l-induced CSF-1 production mechanism, pertussis toxin, which is known to activate adenylate cyclase activity, was used in this experiment. The effect of pertussis toxin on IL-l-induced CSF-1 production is shown in Fig. 3. Pertussis toxin in the concentration range of 10 to 200 rig/ml caused a dose-dependent inhibition of ILl-induced CSF-1 production. At a concentration of 200 rig/ml, it inhibited IL-l-induced CSF-1 production by 70%. To determine whether this inhibitory effect was a specific effect of pertussis toxin on G-protein activity, the ability of benzamide, an ADP-ribosylation inhibitor, to prevent this effect was tested. As shown in Table I, benzamide at 2 mM had no effect on basal CSF-1 production, but abolished the pertussis toxin inhibition of IL-l-stimulated CSF-1 production. These results suggest that the effect of pertussis toxin on the inhibition of IL-l-induced CSF-1 is mediated by the ADP-ribosylation of a Gi component.
6
4
16
Time
20
20
(hrs)
FIG. 2. (a) Time course of IL-l-induced CSF-1 production. Confluent MIA PaCa-2 cells were incubated with IL-1 (7 rig/ml) for 0, 4, 8, 16, and 20 h or cells were incubated for 20 h without IL-1 added (control). Following incubation, media were harvested and assayed for CSF-1 activity in duplicate as described under Materials and Methods. (b) Northern analysis of CSF-1 mRNA. MIA PaCa-2 cells in serum-free DME media were incubated with IL-k (6.8 rig/ml). Cells were harvested at different times and RNA was isolated. Northern analysis of CSF-1 mRNA is described under Materials and Methods. Lane 1,O time; Lane 2,3 h; and Lane 3,8 h.
Inhibition of IL-l-Induced Cholera Toxin
CSF-1 Production
by
Similarly, 50 rig/ml cholera toxin inhibits IL-l-induced CSF-1 production to the control level (Table II) by catalyzing the ADP-ribosylation of G, and activating adenylate cyclase. MBAMG, which has been shown to inhibit cholera toxin-induced ADP-ribosylation (13) at 5 PM has
80 07 w
5
60
: b$ 40 d 2 20
n”
c CONC.
0
10
OF PERTUSSIS
100 TOXIN
200 (nglml)
FIG. 3. Inhibition of IL-l-induced CSF-1 production by toxin. Cultured MIA PaCa-2 cells in serum-free DME media cubated with IL-1 (7 rig/ml) and different concentrations of toxin for 72 h. The conditioned media were assayed for CSF-1
pertussis were inpertussis activity.
INDUCTION
OF MACROPHAGE
TABLE
Inhibition
COLONY-STIMULATING
FACTOR
BY INTERLEUKIN-1
60
T
I
of the Pertussis Toxin Effect by Benzamide No. of colonies
Effecters
18 67 19 29 17 65
Control IL-l (7 rig/ml) PTX (200 rig/ml) IL-1 + PTX BZ (2 mM) IL-l + PTX + BZ
+_ 2 f 9 + 4 + 2 + 1 f 2
Note. Confluent MIA PaCa-2 cells in serum-free medium were incubated with IL-1 (7 rig/ml), pertussis toxin (200 rig/ml), and benzamide (2 mM) separately or in combination for 3 days. The conditioned media thus prepared were assayed for CSF-1 activity as described under Materials and Methods.
no effect on basal CSF-1 production, but abolishes the inhibitory effect of cholera toxin on IL-l-stimulated CSF1 production. Inhibition of IL-l-Induced Dibutytyl-CAMP
CSF-1 Production
Inhibition of CSF-1 Production Forskolin
by Theophylline
and
Theophylline is a phosphodiesterase inhibitor, and forskolin is an adenylate cyclase stimulator. Both chemicals cause an increase in CAMP levels. The results in Table
TABLE
FIG. 4. Inhibition of IL-l-induced CSF-1 production by dibutyrylCAMP. Cultured cells in serum-free DME media were incubated with or without IL-1 and dibutyryl-CAMP. The conditioned media were assayed for CSF-1 activity on mouse marrow cells. (A) Control, nonstimulated conditioned medium; (B) dibutyryl-CAMP (10-r &treated conditioned medium; (C) IL-1 (7 rig/ml)-treated conditioned medium; and (D) dibutyryl-CAMPand IL-l-treated conditioned medium.
by
In order to address whether IL-l stimulation involves adenylate cyclase regulation as suggested by the previous experiment, the effect of an increasing cellular concentration of CAMP on IL-l-induced CSF-1 production was studied. The cell-permeable dibutyryl-CAMP alone had no effect on basal CSF-1 production, but caused a marked inhibition (50%) of IL-l-induced CSF-1 production (Fig. 4). This result suggests that IL-l-induced CSF-1 production is inhibited by increasing CAMP levels.
Inhibition
45
III show that theophylline and forskolin inhibit CSF-1 production. Theophylline at 0.1 mM inhibited IL-l-induced CSF-1 production by 55%. Forskolin at 3.3 mM inhibited IL-l-induced CSF-1 production by 64%. The results show that increasing CAMP levels by theophylline or forskolin are directly correlated to decreasing CSF-1 production into the conditioned medium. Effect of IL-l
on Intracellular
Since it is suggested that IL-l induction may couple to CAMP levels, it was necessary to measure the effect of IL-l on cellular CAMP levels. The effects of IL-l on basal and PTX or theophylline-stimulated CAMP levels in MIA PaCa-2 cells are shown in Table IV. The results show that IL-l alone did not affect CAMP concentration in 10 min. In a separate experiment, CAMP concentrations were measured at 5, 10, 20, and 30 min after the addition of IL-l. No increase of CAMP up to 30 min was observed
II
of the Cholera Toxin Effect by MBAMG Effecters
Control IL-l (7 rig/ml) Cholera toxin (50 rig/ml) IL-1 + Cholera toxin MBAMG (5 /AM) IL-l + Cholera toxin + MBAMG
CAMP Concentration
No. of colonies 57 168 60 89 57 141
+ + + + f +
2 6 3 5 3 9
Note. Cultured MIA PaCa-2 cells in serum-free DME media were incubated with IL-1 (7 rig/ml), cholera toxin (50 rig/ml), and MBAMG (5 pM) separately or in combination for 3 days. The conditioned media were then assayed for CSF-1 activity as described under Materials and Methods.
TABLE
Inhibition
III
of CSF-1 Production and Forskolin Effecters
IL-1 IL-l + theophylline (0.1 mM) IL-1 + forskolin (3.3 FM)
by Theophylline
No. of colonies 542 3 198 f 14 90 71
Note. MIA PaCa-2 cells in serum-free DME media were incubated with different concentrations of theophylline or forskolin for 18 h, and then media were assayed for CSF-1 activity. Theophylline (0.1 mM) or forskolin (3.3 PM) alone did not affect the basal level of CSF production.
46
KU, TABLE Effect
of IL-l
LIU,
AND
WU TABLE
IV
on CAMP
Effect
Levels
of H-7
and HA1004
on IL-land TPA-Stimulated Production
CSF-1 Effecters
CAMP (pmol/106 cells)
IL-1 TPA Theophylline IL-1 + theophylline Pertussis toxin IL-l + Pertussis toxin
19* 17* 20* 39* 28: 28 + 7 22 + 7
Note. MIA PaCa-2 cells (3 X 10’) in 60-mm culture dishes were treated with 1 mM theophylline, 1 mM 3-isobutyl-1-methylxanthine, or 200 ng/ ml pertussis toxin at 37°C for 10 min prior to addition of 7 rig/ml IL1 and/or other effecters. Thirty minutes after addition of effecters, 0.5 ml of 10% TCA was added to terminate the reaction and aqueous samples were extracted with 10 vol of diethyl ether. Then the radioimmunoassay of the CAMP was carried out as described under Materials and Methods. * Standard deviation is less than 1.
(data not shown). These data suggest that the mechanism of action of IL-l-induced CSF-1 production was probably not mediated by activation of adenylate cyclase. The addition of PTX or theophylline to intact cells results in increased 1.5 to 2-fold intracellular CAMP concentrations, as expected, and the increases of CAMP correlate to the decreases in CSF-1 production as shown in Table III. Effect of H-7 and HA1004 on IL-lCSF Production
and TPA-Induced
Since the above results indicated that CAMP levels are not affected by IL-l, the signal pathway induced by IL-l may not involve the activation of adenylate cyclase. The possible involvement of protein kinase C was then investigated. Table V shows that H-7 or HA1004 at 25 PM had no effect on IL-l-induced CSF-1 production. However, H-7 at the same concentration inhibited the TPA (10 ng/ ml)-induced CSF-1 production to the control level. HA1004, which has a higher Ki (40 PM) than H-7 (6 PM) to PKC (14), does not have an obvious effect on TPAinduced CSF-1 production. These results suggest that TPA-induced CSF-1 production is mediated through a pathway involving PKC stimulation, as expected. However, PKC stimulation is not directly involved in IL-linduced CSF-1 production.
V
Effecters
No. of colonies
Control H-7 HA1004 IL-1 IL-1 + H-7 IL-1 + HA1004 TPA TPA + H-7 TPA + HA1004
64f 7 71+ 1 6Ok 4 144 f 11 165 + 22 136 + 4 119f 8 70+ 4 123 k 7
Note. Confluent MIA PaCa-2 cells in serum-free DME media were incubated with 25 HIM H-7 or HA1004 for 2 h, before IL-1 or TPA (10 rig/ml) was added. After 18 h of incubation, the medium was harvested and assayed for CSF-1 activity.
As shown in Fig. V, IL-l treatment of MIA PaCa-2 cells triggered the transient expression of both c-jun and c-fos mRNA earlier than CSF-1 gene expression. The expression level of c-fos reached maximum at 30 min and decreased back to basal level within 3 h (Fig. 5A). However, c-jun expression remains after 3 h with the maximum level at 30 min (Fig. 5A). Their basal level expression is shown in Fig. 5A, Lane 1, and Fig. 5B, Lane 1, respectively. DISCUSSION In the present study, the results show that IL-l can stimulate CSF-1 production in MIA PaCa-2 cells (Fig.
B.
A. IL-1
-
+
+
IL-1
-
+
+
hr.
- .5
3
hr.
-
.5
3
28s 18s
IL-l -Induced c-fos and c-jun Expression Results from the above experiments have shown that adenylate cyclase and PKC are probably not involved in IL-l-induced signal transduction. Since protooncogenes such as c-fos and c-jun expression have been implicated in the regulation of cytokine gene expression (16), their expression in IL-l-induced CSF-1 expression was studied.
123 FIG. 5. Northern blot analysis of MIA PaCa-2 cell RNA. Total preparations from control or IL-l-treated cells were analyzed electrophoresis and hybridized with probe c-fos (A) or c-jun (B) scribed under Materials and Methods. Lane 1, control; Lanes 2 RNA from IL-l-treated cells for 0.5 and 3 h, respectively.
RNA by gel as deand 3,
INDUCTION
OF MACROPHAGE
COLONY-STIMULATING
2a), as in other types of cells reported previously. The concentration of IL-l that we used was similar to the reported value in inducing CSF production in endothelial cell culture (5). By mouse marrow assay, release of CSF1 was stimulated progressively in MIA PaCa-2 cells conditioned by increasing concentrations of IL-l (O-700 ng/ ml). At optimal concentrations of IL-l, detectable CSF1 levels were found between 0 and 4 h of incubation (Fig. 1) and continued to accumulate in the medium over 20 h. Northern analysis (Fig. 2b) of IL-l-induced CSF-1 mRNA levels showed correlations consistent with biological activity (Fig. 1). These data indicate that IL-l stimulates CSF-1 mRNA transcription and protein production in MIA PaCa-2 cells. Although the mechanism is not well understood, results from this experiment suggest that IL-l-induced CSF-1 expression did not involve adenylate cyclase activation. Treatment with either pertussis toxin or cholera toxin led to the increase of CAMP and consequently caused the inhibition of CSF-1 production. The effect by these toxins can be reversed by their respective inhibitors of ADPribosylation, benzamide at 2 mM for pertussis toxin (Table I) and MBAMB at 5 PM for cholera toxin (Table II). These results suggest that the effect of pertussis toxin and cholera toxin on the inhibition of IL-l-induced CSF-1 production are mediated by the ADP-ribosylation of Gi and G,, leading to the activation of adenylate cyclase. The addition of theophylline, forskolin, and dibutyl-CAMP ( 10e7 M) to the cell culture also inhibited the IL-l-induced CSF-1 production (Fig. 4 and Table III). These results demonstrated further that an increase of CAMP levels inhibited CSF-1 expression. Furthermore, measurement of CAMP concentration shows that IL-l alone did not affect CAMP concentration. These data suggested the mechanism of action of IL-l-induced CSF-1 production was probably not mediated by adenylate cyclase. If IL-l-induced CSF-1 expression is not mediated through CAMP, then what is the other possible pathway? TPA has been used as an inducer besides IL-l for CSF production. The data show CSF-1 production stimulated by TPA can be inhibited by the specific PKC inhibitor H-7, indicating the involvement of PKC in the TPA-induced CSF-1 system. However, H-7 shows no effect on IL-l-induced CSF-1 synthesis, indicating that the signaling pathway is PKC independent (Table V). Results from these preliminary studies suggest that IL-l- and TPA-induced CSF-1 syntheses are mediated by different signal transduction pathways. Yamato et al. (15) similarly reported that stimulation of PKC dramatically increased levels of GM-CSF mRNA; however, blockage of PKC activity did not attenuate accumulation of GM-CSF mRNA which are stimulated by TNF and NaF. Results from the above experiments have shown that the signal transduction mechanism is not mediated by adenylate cyclase nor protein kinase C pathways. The identity of the second
FACTOR
BY INTERLEUKIN-1
47
messenger of signal transduction in IL-l-induced CSF-1 expression remains to be investigated. Since protooncogenes such as c-fos and c-jun expression have been implicated in the regulation of cytokine gene expressions, expression in IL-l-induced MIA PaCa-2 cells has been studied. Muegge et al. (16) showed that IL-linduced T-lymphocyte growth, protooncogen c-jun acting as a second signal (together with antigen), enhances the production of IL-2. One of the IL-l-responsive elements in the promoter region of the human IL-2 gene was similar to the binding site for the transcription factor AP-1. IL1 enhanced expression of c-jun mRNA, whereas the antigenic signal enhanced mRNA expression of c-fos. The c-fos gene encodes a nuclear protein which participates in the formation of the AP-1 transcription factor complex (17). A major component of this complex is the product of the c-jun protooncogen (17). TPA in this system also enhanced c-fos expression as the initial signal. Thus, the two components of the AP-1 factor are independently regulated, and IL-l and TPA may stimulate CSF gene expression through different signals which interact with different regulatory elements in the promoter region. One consistent result from the experiment on IL-l-induced CSF-1 expression was that c-fos and c-jun expression preceded the increase of the CSF-1 mRNA level. The expression of c-fos occurred 30 min after IL-l induction and decreased to an undetectable level after 3 h, while the expression of c-jun also occurred 30 min after IL-l induction but lasted more than 3 h. Although the present results do not provide any data to explain the role of cjun and c-fos in activation of CSF-1 expression or the signal transduction mechanism in IL-l-induced CSF-1 expression, the induced expression by IL-l of early genes such as c-fos and c-jun is strongly suggestive that expression of both protooncogenes leads to CSF-1 expression similar to IL-2 expression (16). Recently, we have also observed that the expression of c-fos and c-jun preceded CSF-1 production in TPA-induced U-937 cells (18). An increase in the CAMP level, on the other hand, can stimulate the phosphorylation of c-jun or c-fos (19), resulting in a loss of their binding to DNA and the inactivation of CSF-1 gene expression. In summary, the present study has shown that IL-1 can stimulate CSF-1 expression in MIA PaCa-2 cells. The induction does not involve the elevation of CAMP and the signal pathway appears to be different from the induction by TPA. However, an increase in the CAMP level causes inhibition of CSF-1 expression. The molecular mechanism of the regulation of CSF-1 expression remains to be further investigated. ACKNOWLEDGMENTS This work was supported by Robert A. Welch Foundation Grant B1058, USPHS Grants AM 31624 and BRSG RR-05879, and a faculty research grant from the University of North Texas. The authors thank
48
KU,
LIU,
Dr. Richard A. Easom for review and Mrs. Margie Schluter for preparation of the manuscript.
REFERENCES 1. Clark, S. C., and Kamen, R. (1987) Science 236, 1229-1237. 2. Nicola, N. A. (1989) Annu. Z&w. Biochem. 58, 45-78. 3. Zucali, J. R., Dinarello, C. A., Oblon, D. J., Gross, M. A., Anderson, L., and Weiner, R. A. (1986) J. Clin. Znuest. 77, 1857-1863. 4. Segal, G. M., McCall, E., Steve, T., and Bagby, G. C. (1987) J. Zmmunol. 138,1772-1778. 5. Zsebo, K. M., Yuschenkoff, V. N., Schiffer, S., Chang, D., McCall, E., Dinarello, C. A., Brown, M. A., Altrock, B., and Bagby, G. C. (1988) Blood 71,99-103. 6. Ralph, P., Warren, M. K., Lee, M. T., Csejtey, J., Weaver, J. F., Broxmeyer, H. E., Williams, D. E., Stanley, E. R., and Kawasaki, E. S. (1986) Blood 68, 633-639. Yunis, A. A., Arimura, G. K., and Russin, D. J. (1977) Znt. J. Cancer
19,128-134. Wu, M.-C., Cini, J. K., and Yunis, A. A. (1979) J. Biol. Chem. 254,
6226-6228. Shieh, J.-H., Cini, J. K., Wu, M.-C., and Yunis, A. A. (1989) Arch. Biochem. Biophys. 253, 205-213.
AND
WU
10. Kawasaki, E. S., Lander, M. B., Wang, A. M., Van Arsdell, J., Warren, M. K., Coyne, M. Y., Schwickart, D. L., Lee, M., Wilson, K. J., Boosman, A., Stanley, E. R., Ralph, P., and Mark, D. F. (1985) Science 230,291-296. 11. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 12. Hopkins,
N. K., and Gorman, R. R. (1981) J. Clin. Znuest. 67,540-
546. 13. Tait, R. M., and Nassau, P. M. (1984) Eur. J. Biochem. 143,213. 14. Asano, T., and Hidaka,
H. (1984) J. Pharmacol.
Exp. Ther. 231,
141. 15. Yamato, K., Ei-Hajjaoui, Blood 74, 1314-1320.
Z., Kuo, J. F., and Koeffler,
H. P. (1989)
16. Muegge, K., Williams, T. M., Kant, J., Karin, M., Chiu, R., Schmidt, A., Siebenlist, U., Young, H. A., and Durum, S. K. (1989) Science
246,249. 17. Rauscher, F. J., III, Cohen, D. R., Curran, T., Bos, T. J., Vogt, P. K., Bohmann,
D., Tjian, R., and Franza, B. R., Jr. (1988) Science
240,lOlO. 18. Liu, M. Y., and Wu, M. C. (1991) unpublished 19. Woodgett, J. R. (1991) TZBS 16, 177-181.
results.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 295, No. 1, May 15, pp. 42-48, 1992
Stimulation of Macrophage Synthesis by Interleukin-I
Colony-Stimulating
Jean C.-Y. Ku, Mu-Ya Liu, and Ming-chi
Wul
Factor
Department of Biochemistry and Molecular Biolozy, Texas College of Osteopathic Medicine/ Urkersity of North Texas-Fort Worth, Texas 76107
Received November
12, 1991, and in revised form January
17, 1992
Interleukin-1 (IL-l), which plays an important role in the inflammatory response, was found to induce colonystimulating factor-l (CSF-1) expression in the MIA PaCa2 cells. IL- l-induced CSF- 1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-l-induced CSF1 production was inhibited by cholera toxin and this inhibition was reversed by an a&nine analog, pmethoxybenzylaminodecamethylene guanidine sulfate. DibutyrylCAMP as well as other CAMP elevating agents such as theophylline and forskolin also suppressed IL-l-induced CSF-1 production, suggesting that CAMP concentrations inversely regulate the biosynthesis of CSF- 1. Measurement of CAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the CAMP level. IL-linduced CSF- 1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12-0-tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL- 1 -induced CSF- 1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF- 1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression. 0 1992 Apress. Inc.
Although the production of CSFs2 by recombinant DNA techniques has been accomplished and CSFs in general 1 To whom correspondence should be addressed. ’ Abbreviations used: CSF, colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-l, interleukin-1; TPA, 12-0-tetradecanoylphorbol-13-acetate; DME, Dulbecco’s modified Eagle’s; PBS, phosphate-buffered saline; SSC, standard saline citrate; SDS, sodium dodecyl sulfate; MBAMG, p-methoxybenzylaminodecamethylene guanidine sulfate; RIA, radioimmunoassay; TCA, trichloroacetic acid, DMSO, dimethyl sulfoxide; PKC, protein kinase C; TNF, 2,4,7-trinitrofluorenone; PTX, pertussis toxin; BZ, benzamide. 42
have been reviewed recently (1, 2), the molecular mechanisms involved in the regulation of their synthesis have not been investigated systematically. A variety of cell types such as macrophages, T-lymphocytes, endothelial cells, and fibroblasts can produce CSFs, particularly under cytokine stimulation (3-5). Bacterial endotoxin has long been used as the stimulator for CSF production. Other mitogens have also been shown to stimulate CSF production. IL-l is a member of the monokine family produced primarily by monocytes. It is a key mediator of the host response to infections, inflammations, and immunologic challenges. Monocytes, T-lymphocytes, B-lymphocytes, hepatocytes, neutrophils, fibroblasts, and endothelial cells can be stimulated by IL-l to generate many features of the inflammatory reaction (3-5). The effects of acute and chronic inflammation on the hematopoietic system include release of neutrophils from the vascular marginal pool; premature release of neutrophils from the marrow into the circulation; enhanced neutrophil chemotaxis to, and activation at, sites of inflammation; and a sustained increase in neutrophil and monocyte production. Many of these responses are evoked by the various CSFs in vitro. Zucali et al. (3) have shown that IL-l-stimulated fibroblasts produce GM-CSF and prostaglandin E2. Segal et al. (4) and Zsebo et al. (5) have reported that ILl-stimulated endothelial cells release multilineage human CSF activity in addition to GM-CSF as previously reported. Ralph et al. (6) have also reported that TPA stimulated CSF-1 production from MIA PaCa-2 cells and that the increase of CSF-1 production is correlated with mRNA expression. However, the mechanism involved in the regulation of CSF production stimulated by monokines and other stimuli is not very well understood. Furthermore, whether the regulation is at the transcriptional or translational level remains to be studied. The human pancreatic carcinoma cell line MIA PaCa2 established by Yunis et al. (7) has been a rich source for CSF-1 (8, 9). This cell line has been used to clone CSF-1 cDNA (10) and is used in this experiment to study 0003-9S61/92
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
INDUCTION
OF MACROPHAGE
COLONY-STIMULATING
the mechanism involved in the stimulation of CSF-1 production by IL-l. Data presented in this article indicate that CSF-1 synthesis is inversely regulated by CAMP levels. Although the role of the IL-l-induced signal transduction pathway in enhancing CSF-1 expression is still not clear, the involvement of protooncogene c-fos and cjun expression is suggested. MATERIALS
AND
METHODS
Cell culture. The MIA PaCa-2 cells were cultured in DME (Dulbecco’s modified Eagle’s) medium and supplemented with 5% fetal calf serum, 3% newborn calf serum, penicillin (100 pg/ml), and streptomycin (100 ag/ml) as described previously (8). The confluent cultures were changed to serum-free DME medium. Different concentrations of lymphokine and other modulators were added to the culture. The incubation was then continued for different periods of time, depending on the culture conditions of each experiment. The conditioned medium thus prepared was assayed for CSF-1 activity as described below. Mouse marrow assay for CSF. The conditioned media prepared under different stimuli were assayed for CSF-1 activity using the mouse bone marrow method (9). Mouse bone nucleated cells (10s) obtained from C57 BL/6 mice (Jackson Laboratories, Bar Harbor) were added to a final volume of 1.2 ml soft-agar medium with 50 al of conditioned medium. The agar cultures were then incubated at 37°C under 6% CO, for 5 days. Aggregates of 50 or more cells were counted as colonies using a dissecting microscope. RNA isolation and Northern blot analysis. Confluent MIA PaCa-2 cells were cultured with or without IL-1 for various times. The cells were washed twice with phosphate-buffered saline (PBS). Total RNA (20 pg) extracted by the guanidium/cesium chloride method (11) was denatured and size-fractionated by 1% formaldehyde-agarose gel electrophoresis and transferred to a nylon membrane by capillary blotting using 20X SSC. For the equality of RNA loading, parallel RNA samples were stained with ethidium bromide to visualize 28s and 18s ribosomal RNA bonds. The membrane was irradiated with uv light for 5 min. Prehybridization was performed in 5X SSC, 15X Denhardt’s, 0.1% SDS, and denatured salmon sperm DNA at 65°C for 18 h. The blot was hybridized with y-32P-labeled 40-mer synthetic probe (CSF-1, c-fos, and c-jun) purchased from Oncogene Science, Inc. Hybridization was performed in the same buffer for another 18 h at 65°C. Membranes, washed with 1X SSC and 0.1% SDS at room temperature, 42°C and 65°C for half an hour each time, were subjected to autoradiography at -70°C by exposing to Kodak XAR5 film with an intensifying screen. Recombinant IL-I. Recombinant IL-lo (rHu. IL-la [117-2711 Ro245008) (gift from Hoffmann-LaRoche, Inc., Nutley, NJ) was added to the cell cultures in the concentration range of 0.7 to 700 rig/ml. The activity is 2 X 10s U/ml by DlO cell thymidine uptake assay. Effectof IL-l on CSF-1 production. After confluence, growth medium was removed from MIA PaCa-2 cells. The cells were washed with PBS and DME serum-free medium was added. IL-l at concentrations of 0, 0.7, 7.0, 70.0, and 700.0 rig/ml was added to the culture plates and incubated for an additional 72 h. The media were harvested, desalted by PD.10 (Pharmacia), sterilized by filtration, and assayed for CSF-1 activity in triplicate plates. Time course of IL-l-induced CSF-1 production. Confluent MIA PaCa2 cells in DME serum-free media were cultured with IL-l (7 rig/ml) for different periods of time (0, 4, 8, 16, and 20 h) or without IL-l for 20 h as control. The media were harvested, desalted by PD-10, sterilized by filtration, and assayed for CSF-1 activity. Effect of pertussis toxin and cholera toxin on IL-l-induced CSF-1 production. Cultured MIA PaCa-2 cells in serum-free DME medium were incubated with IL-1 (7 rig/ml), pertussis toxin (200 rig/ml, List Biological Laboratories, CA), and benzamide (2 mM) separately or in combination for 72 h. The conditioned media were harvested and assayed for CSF-
FACTOR
BY INTERLEUKIN-1
43
1 activity in triplicate plates. For the cholera toxin experiment, IL-ltreated MIA PaCa-2 cells were incubated with cholera toxin (10 to 1000 pg/ml) and 5 FM MBAMG, as in pertussis toxin experiments. Effectof dibuty$cAMP on IL-l -induced CSF-1 production. Cultured MIA PaCa-2 cells in serum-free DME medium were incubated with or without IL-l (7 rig/ml) and dibutyryl-CAMP (10-r M) for 48 h. The stock solution of dibutyryl-CAMP (10e3 M) was prepared in absolute ethanol. The conditioned media were assayed for CSF-1 activity as described above. Radioimmunoassay of CAMP. The level of CAMP from the CSF-1 stimulation or inhibition study is measured by radioimmunoassay (RIA) as described by Hopkins and Gorman (12). Briefly, cells (3 X 10s) in tissue culture plates are treated with or without 1 mM theophylline at 37’C for 10 min prior to adding effecters. At the end of incubation with the addition of effecters (30 min), 0.5 ml of 10% TCA is added and aqueous samples are extracted three times with 10 vol of diethyl ether. The aqueous phase is evaporated and the residue dissolved into 50 mM sodium acetate buffer, pH 6.2. The RIA of CAMP is carried out as suggested by the manufacturer (NEN Research Products, NEK-033). Effect of theophylline and forsholin on CSF-1 production. Theophylline and forskolin, which can increase the cellular CAMP level by inhibiting phosphodiesterase and stimulating adenylate cyclase, respectively, are used to further study the role of CAMP in regulating CSF-1 synthesis. The stocks of theophylline and forskolin are prepared in 100% DMSO and absolute ethanol, respectively. Confluent MIA PaCa2 cells in serum-free DME media were incubated with 0, 0.01, 0.1, and 0.5 mM of theophylline (Sigma) or 1.65, 3.3, and 6.6 pM of forskolin (Sigma) to a final volume of 2.5 ml. After 18 h incubation, the medium was harvested and assayed for CSF-1 activity. Effect of H-7 and HA1004 on IL-l-induced CSF-1 production. Two protein kinase inhibitors were used in this study to differentiate whether the IL-l-induced CSF-1 production involved the CAMP-dependent protein kinase or protein kinase C process as the signal transduction mechanism. To petri dishes with confluent cultured MIA PaCa-2 cells was added serum-free DME medium, which was then incubated with 25 pM H-7 or HA1004 (Seikagaku America, Inc., St. Petersburg, FL) for 2 h before IL-1 (7 rig/ml) or TPA (10 rig/ml) was added. Incubation was continued for 18 h and the media were harvested and assayed for CSF1 activity. The stock solutions of H-7 and HA1004 were 10 mM in distilled water. Data analysis. Data are presented as the mean + SD with triplicate determination or given assay parameters from separate dishes within each experiment and normalized to concurrent control cultures.
RESULTS
IL-1 -Induced CSF-1 Production As shown in Fig. 1, cultured nonstimulated MIA PaCa2 cells (control) produced low levels of CSF as measured by the mouse marrow soft agar assay. By dose-response analysis, the addition of increasing concentrations of IL1 (0.7 to 700 rig/ml) enhanced the production of CSF-1 in the medium by about threefold. The concentration of IL-l that resulted in maximum stimulation was around 7.0 rig/ml. Therefore, the IL-l concentration of 7.0 ng/ ml was used for the rest of the study. The control experiment was carried out to check if IL-l has any colonystimulating activity alone or synergistic effect on colony formation with CSFs. The result showed that IL-l had no direct effect on colony formation nor any synergistic effect with CSF at these concentrations (data not shown). As shown in Fig. 2a, IL-l-induced CSF-1 activity was first detected between 0 and 4 h, and continued to accu-
44
KU,
LIU,
AND
WU
200
Y
b
IL-1
-
+
+
hr
-
.5
3
1
2
3
150
z 60
100
3 s
50
0
0
0.7
7
CONC.
OF IL-I
70
700
0
(rig/ml)
FIG. 1. Stimulation of CSF-1 production by IL-l. MIA PaCa-2 cells were cultured until confluence. The growth medium was removed, the cells were washed with PBS, and serum-free medium was added. IL-1 at different concentrations was then added and the incubation was continued for 72 h. The media were harvested, desalted, sterilized by filtration, and assayed for CSF-1 activity in triplicate plates.
mulate in the medium over 20 h. A fourfold stimulation over the control was observed at 20 h. By Northern blot analysis, IL-l also increased accumulation of CSF-1 mRNA levels after 3 h treatment (Fig. 2b, lane 3), compared with the control which was without IL-l treatment (Fig. 2b, Lane 1). The increase of CSF-1 activity in the medium is consistent with the increase of mRNA as induced by IL-l, suggesting the stimulation of CSF-1 expression at the transcriptional level.
Inhibition of IL-l-Induced Pertussis Toxin
CSF-1 Production
by
In order to examine whether G-proteins are involved in the IL-l-induced CSF-1 production mechanism, pertussis toxin, which is known to activate adenylate cyclase activity, was used in this experiment. The effect of pertussis toxin on IL-l-induced CSF-1 production is shown in Fig. 3. Pertussis toxin in the concentration range of 10 to 200 rig/ml caused a dose-dependent inhibition of ILl-induced CSF-1 production. At a concentration of 200 rig/ml, it inhibited IL-l-induced CSF-1 production by 70%. To determine whether this inhibitory effect was a specific effect of pertussis toxin on G-protein activity, the ability of benzamide, an ADP-ribosylation inhibitor, to prevent this effect was tested. As shown in Table I, benzamide at 2 mM had no effect on basal CSF-1 production, but abolished the pertussis toxin inhibition of IL-l-stimulated CSF-1 production. These results suggest that the effect of pertussis toxin on the inhibition of IL-l-induced CSF-1 is mediated by the ADP-ribosylation of a Gi component.
6
4
16
Time
20
20
(hrs)
FIG. 2. (a) Time course of IL-l-induced CSF-1 production. Confluent MIA PaCa-2 cells were incubated with IL-1 (7 rig/ml) for 0, 4, 8, 16, and 20 h or cells were incubated for 20 h without IL-1 added (control). Following incubation, media were harvested and assayed for CSF-1 activity in duplicate as described under Materials and Methods. (b) Northern analysis of CSF-1 mRNA. MIA PaCa-2 cells in serum-free DME media were incubated with IL-k (6.8 rig/ml). Cells were harvested at different times and RNA was isolated. Northern analysis of CSF-1 mRNA is described under Materials and Methods. Lane 1,O time; Lane 2,3 h; and Lane 3,8 h.
Inhibition of IL-l-Induced Cholera Toxin
CSF-1 Production
by
Similarly, 50 rig/ml cholera toxin inhibits IL-l-induced CSF-1 production to the control level (Table II) by catalyzing the ADP-ribosylation of G, and activating adenylate cyclase. MBAMG, which has been shown to inhibit cholera toxin-induced ADP-ribosylation (13) at 5 PM has
80 07 w
5
60
: b$ 40 d 2 20
n”
c CONC.
0
10
OF PERTUSSIS
100 TOXIN
200 (nglml)
FIG. 3. Inhibition of IL-l-induced CSF-1 production by toxin. Cultured MIA PaCa-2 cells in serum-free DME media cubated with IL-1 (7 rig/ml) and different concentrations of toxin for 72 h. The conditioned media were assayed for CSF-1
pertussis were inpertussis activity.
INDUCTION
OF MACROPHAGE
TABLE
Inhibition
COLONY-STIMULATING
FACTOR
BY INTERLEUKIN-1
60
T
I
of the Pertussis Toxin Effect by Benzamide No. of colonies
Effecters
18 67 19 29 17 65
Control IL-l (7 rig/ml) PTX (200 rig/ml) IL-1 + PTX BZ (2 mM) IL-l + PTX + BZ
+_ 2 f 9 + 4 + 2 + 1 f 2
Note. Confluent MIA PaCa-2 cells in serum-free medium were incubated with IL-1 (7 rig/ml), pertussis toxin (200 rig/ml), and benzamide (2 mM) separately or in combination for 3 days. The conditioned media thus prepared were assayed for CSF-1 activity as described under Materials and Methods.
no effect on basal CSF-1 production, but abolishes the inhibitory effect of cholera toxin on IL-l-stimulated CSF1 production. Inhibition of IL-l-Induced Dibutytyl-CAMP
CSF-1 Production
Inhibition of CSF-1 Production Forskolin
by Theophylline
and
Theophylline is a phosphodiesterase inhibitor, and forskolin is an adenylate cyclase stimulator. Both chemicals cause an increase in CAMP levels. The results in Table
TABLE
FIG. 4. Inhibition of IL-l-induced CSF-1 production by dibutyrylCAMP. Cultured cells in serum-free DME media were incubated with or without IL-1 and dibutyryl-CAMP. The conditioned media were assayed for CSF-1 activity on mouse marrow cells. (A) Control, nonstimulated conditioned medium; (B) dibutyryl-CAMP (10-r &treated conditioned medium; (C) IL-1 (7 rig/ml)-treated conditioned medium; and (D) dibutyryl-CAMPand IL-l-treated conditioned medium.
by
In order to address whether IL-l stimulation involves adenylate cyclase regulation as suggested by the previous experiment, the effect of an increasing cellular concentration of CAMP on IL-l-induced CSF-1 production was studied. The cell-permeable dibutyryl-CAMP alone had no effect on basal CSF-1 production, but caused a marked inhibition (50%) of IL-l-induced CSF-1 production (Fig. 4). This result suggests that IL-l-induced CSF-1 production is inhibited by increasing CAMP levels.
Inhibition
45
III show that theophylline and forskolin inhibit CSF-1 production. Theophylline at 0.1 mM inhibited IL-l-induced CSF-1 production by 55%. Forskolin at 3.3 mM inhibited IL-l-induced CSF-1 production by 64%. The results show that increasing CAMP levels by theophylline or forskolin are directly correlated to decreasing CSF-1 production into the conditioned medium. Effect of IL-l
on Intracellular
Since it is suggested that IL-l induction may couple to CAMP levels, it was necessary to measure the effect of IL-l on cellular CAMP levels. The effects of IL-l on basal and PTX or theophylline-stimulated CAMP levels in MIA PaCa-2 cells are shown in Table IV. The results show that IL-l alone did not affect CAMP concentration in 10 min. In a separate experiment, CAMP concentrations were measured at 5, 10, 20, and 30 min after the addition of IL-l. No increase of CAMP up to 30 min was observed
II
of the Cholera Toxin Effect by MBAMG Effecters
Control IL-l (7 rig/ml) Cholera toxin (50 rig/ml) IL-1 + Cholera toxin MBAMG (5 /AM) IL-l + Cholera toxin + MBAMG
CAMP Concentration
No. of colonies 57 168 60 89 57 141
+ + + + f +
2 6 3 5 3 9
Note. Cultured MIA PaCa-2 cells in serum-free DME media were incubated with IL-1 (7 rig/ml), cholera toxin (50 rig/ml), and MBAMG (5 pM) separately or in combination for 3 days. The conditioned media were then assayed for CSF-1 activity as described under Materials and Methods.
TABLE
Inhibition
III
of CSF-1 Production and Forskolin Effecters
IL-1 IL-l + theophylline (0.1 mM) IL-1 + forskolin (3.3 FM)
by Theophylline
No. of colonies 542 3 198 f 14 90 71
Note. MIA PaCa-2 cells in serum-free DME media were incubated with different concentrations of theophylline or forskolin for 18 h, and then media were assayed for CSF-1 activity. Theophylline (0.1 mM) or forskolin (3.3 PM) alone did not affect the basal level of CSF production.
46
KU, TABLE Effect
of IL-l
LIU,
AND
WU TABLE
IV
on CAMP
Effect
Levels
of H-7
and HA1004
on IL-land TPA-Stimulated Production
CSF-1 Effecters
CAMP (pmol/106 cells)
IL-1 TPA Theophylline IL-1 + theophylline Pertussis toxin IL-l + Pertussis toxin
19* 17* 20* 39* 28: 28 + 7 22 + 7
Note. MIA PaCa-2 cells (3 X 10’) in 60-mm culture dishes were treated with 1 mM theophylline, 1 mM 3-isobutyl-1-methylxanthine, or 200 ng/ ml pertussis toxin at 37°C for 10 min prior to addition of 7 rig/ml IL1 and/or other effecters. Thirty minutes after addition of effecters, 0.5 ml of 10% TCA was added to terminate the reaction and aqueous samples were extracted with 10 vol of diethyl ether. Then the radioimmunoassay of the CAMP was carried out as described under Materials and Methods. * Standard deviation is less than 1.
(data not shown). These data suggest that the mechanism of action of IL-l-induced CSF-1 production was probably not mediated by activation of adenylate cyclase. The addition of PTX or theophylline to intact cells results in increased 1.5 to 2-fold intracellular CAMP concentrations, as expected, and the increases of CAMP correlate to the decreases in CSF-1 production as shown in Table III. Effect of H-7 and HA1004 on IL-lCSF Production
and TPA-Induced
Since the above results indicated that CAMP levels are not affected by IL-l, the signal pathway induced by IL-l may not involve the activation of adenylate cyclase. The possible involvement of protein kinase C was then investigated. Table V shows that H-7 or HA1004 at 25 PM had no effect on IL-l-induced CSF-1 production. However, H-7 at the same concentration inhibited the TPA (10 ng/ ml)-induced CSF-1 production to the control level. HA1004, which has a higher Ki (40 PM) than H-7 (6 PM) to PKC (14), does not have an obvious effect on TPAinduced CSF-1 production. These results suggest that TPA-induced CSF-1 production is mediated through a pathway involving PKC stimulation, as expected. However, PKC stimulation is not directly involved in IL-linduced CSF-1 production.
V
Effecters
No. of colonies
Control H-7 HA1004 IL-1 IL-1 + H-7 IL-1 + HA1004 TPA TPA + H-7 TPA + HA1004
64f 7 71+ 1 6Ok 4 144 f 11 165 + 22 136 + 4 119f 8 70+ 4 123 k 7
Note. Confluent MIA PaCa-2 cells in serum-free DME media were incubated with 25 HIM H-7 or HA1004 for 2 h, before IL-1 or TPA (10 rig/ml) was added. After 18 h of incubation, the medium was harvested and assayed for CSF-1 activity.
As shown in Fig. V, IL-l treatment of MIA PaCa-2 cells triggered the transient expression of both c-jun and c-fos mRNA earlier than CSF-1 gene expression. The expression level of c-fos reached maximum at 30 min and decreased back to basal level within 3 h (Fig. 5A). However, c-jun expression remains after 3 h with the maximum level at 30 min (Fig. 5A). Their basal level expression is shown in Fig. 5A, Lane 1, and Fig. 5B, Lane 1, respectively. DISCUSSION In the present study, the results show that IL-l can stimulate CSF-1 production in MIA PaCa-2 cells (Fig.
B.
A. IL-1
-
+
+
IL-1
-
+
+
hr.
- .5
3
hr.
-
.5
3
28s 18s
IL-l -Induced c-fos and c-jun Expression Results from the above experiments have shown that adenylate cyclase and PKC are probably not involved in IL-l-induced signal transduction. Since protooncogenes such as c-fos and c-jun expression have been implicated in the regulation of cytokine gene expression (16), their expression in IL-l-induced CSF-1 expression was studied.
123 FIG. 5. Northern blot analysis of MIA PaCa-2 cell RNA. Total preparations from control or IL-l-treated cells were analyzed electrophoresis and hybridized with probe c-fos (A) or c-jun (B) scribed under Materials and Methods. Lane 1, control; Lanes 2 RNA from IL-l-treated cells for 0.5 and 3 h, respectively.
RNA by gel as deand 3,
INDUCTION
OF MACROPHAGE
COLONY-STIMULATING
2a), as in other types of cells reported previously. The concentration of IL-l that we used was similar to the reported value in inducing CSF production in endothelial cell culture (5). By mouse marrow assay, release of CSF1 was stimulated progressively in MIA PaCa-2 cells conditioned by increasing concentrations of IL-l (O-700 ng/ ml). At optimal concentrations of IL-l, detectable CSF1 levels were found between 0 and 4 h of incubation (Fig. 1) and continued to accumulate in the medium over 20 h. Northern analysis (Fig. 2b) of IL-l-induced CSF-1 mRNA levels showed correlations consistent with biological activity (Fig. 1). These data indicate that IL-l stimulates CSF-1 mRNA transcription and protein production in MIA PaCa-2 cells. Although the mechanism is not well understood, results from this experiment suggest that IL-l-induced CSF-1 expression did not involve adenylate cyclase activation. Treatment with either pertussis toxin or cholera toxin led to the increase of CAMP and consequently caused the inhibition of CSF-1 production. The effect by these toxins can be reversed by their respective inhibitors of ADPribosylation, benzamide at 2 mM for pertussis toxin (Table I) and MBAMB at 5 PM for cholera toxin (Table II). These results suggest that the effect of pertussis toxin and cholera toxin on the inhibition of IL-l-induced CSF-1 production are mediated by the ADP-ribosylation of Gi and G,, leading to the activation of adenylate cyclase. The addition of theophylline, forskolin, and dibutyl-CAMP ( 10e7 M) to the cell culture also inhibited the IL-l-induced CSF-1 production (Fig. 4 and Table III). These results demonstrated further that an increase of CAMP levels inhibited CSF-1 expression. Furthermore, measurement of CAMP concentration shows that IL-l alone did not affect CAMP concentration. These data suggested the mechanism of action of IL-l-induced CSF-1 production was probably not mediated by adenylate cyclase. If IL-l-induced CSF-1 expression is not mediated through CAMP, then what is the other possible pathway? TPA has been used as an inducer besides IL-l for CSF production. The data show CSF-1 production stimulated by TPA can be inhibited by the specific PKC inhibitor H-7, indicating the involvement of PKC in the TPA-induced CSF-1 system. However, H-7 shows no effect on IL-l-induced CSF-1 synthesis, indicating that the signaling pathway is PKC independent (Table V). Results from these preliminary studies suggest that IL-l- and TPA-induced CSF-1 syntheses are mediated by different signal transduction pathways. Yamato et al. (15) similarly reported that stimulation of PKC dramatically increased levels of GM-CSF mRNA; however, blockage of PKC activity did not attenuate accumulation of GM-CSF mRNA which are stimulated by TNF and NaF. Results from the above experiments have shown that the signal transduction mechanism is not mediated by adenylate cyclase nor protein kinase C pathways. The identity of the second
FACTOR
BY INTERLEUKIN-1
47
messenger of signal transduction in IL-l-induced CSF-1 expression remains to be investigated. Since protooncogenes such as c-fos and c-jun expression have been implicated in the regulation of cytokine gene expressions, expression in IL-l-induced MIA PaCa-2 cells has been studied. Muegge et al. (16) showed that IL-linduced T-lymphocyte growth, protooncogen c-jun acting as a second signal (together with antigen), enhances the production of IL-2. One of the IL-l-responsive elements in the promoter region of the human IL-2 gene was similar to the binding site for the transcription factor AP-1. IL1 enhanced expression of c-jun mRNA, whereas the antigenic signal enhanced mRNA expression of c-fos. The c-fos gene encodes a nuclear protein which participates in the formation of the AP-1 transcription factor complex (17). A major component of this complex is the product of the c-jun protooncogen (17). TPA in this system also enhanced c-fos expression as the initial signal. Thus, the two components of the AP-1 factor are independently regulated, and IL-l and TPA may stimulate CSF gene expression through different signals which interact with different regulatory elements in the promoter region. One consistent result from the experiment on IL-l-induced CSF-1 expression was that c-fos and c-jun expression preceded the increase of the CSF-1 mRNA level. The expression of c-fos occurred 30 min after IL-l induction and decreased to an undetectable level after 3 h, while the expression of c-jun also occurred 30 min after IL-l induction but lasted more than 3 h. Although the present results do not provide any data to explain the role of cjun and c-fos in activation of CSF-1 expression or the signal transduction mechanism in IL-l-induced CSF-1 expression, the induced expression by IL-l of early genes such as c-fos and c-jun is strongly suggestive that expression of both protooncogenes leads to CSF-1 expression similar to IL-2 expression (16). Recently, we have also observed that the expression of c-fos and c-jun preceded CSF-1 production in TPA-induced U-937 cells (18). An increase in the CAMP level, on the other hand, can stimulate the phosphorylation of c-jun or c-fos (19), resulting in a loss of their binding to DNA and the inactivation of CSF-1 gene expression. In summary, the present study has shown that IL-1 can stimulate CSF-1 expression in MIA PaCa-2 cells. The induction does not involve the elevation of CAMP and the signal pathway appears to be different from the induction by TPA. However, an increase in the CAMP level causes inhibition of CSF-1 expression. The molecular mechanism of the regulation of CSF-1 expression remains to be further investigated. ACKNOWLEDGMENTS This work was supported by Robert A. Welch Foundation Grant B1058, USPHS Grants AM 31624 and BRSG RR-05879, and a faculty research grant from the University of North Texas. The authors thank
48
KU,
LIU,
Dr. Richard A. Easom for review and Mrs. Margie Schluter for preparation of the manuscript.
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