post-transcriptional control of il-1 gene expression in the acute monocytic leukemia line thp-1

Vol. 156, No. 2, 1988

October 31, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 830-839

POST-TRANSCRIPTIONAL CONTROL OF I L - 1 GENE EXPRESSION IN THE ACUTE MONOCYTIC LEUKEMIA LINE THP-1

MARTIN TURNER, DAVID CHANTRY, AND MARC FELDMANN

Chafing Cross Sunley Research Centre, Lurgan Avenue, London, W6 8LW

United Kingdom

Received September 7, 1988

The acute monocytic leukemia cell line THP- I secretes predominantly IL-I~ after treatment with bacterial lipopolysaccharide and tumour promoting phorbol ester (PMA). IL-I~ is also secreted, but represents less than 10% of the total IL-I activity. This differential is reflected at the level of mRNA as IL-18 mRNA is more abundant than IL-la mRNA. Studies of transcription in isolated nuclei however indicate that each gene is transcribed at a similar rate, suggesting that post-transcriptional mechanisms regulate the relative abundance of IL-la and IL-18 mRNA. Measurement of RNA half life after addition of a-amanitin (an inhibitor of RNA polymerase II) indicate that IL-I~ mRNA is not as stab]e as IL-18 mRNA suggesting one mechanism for tile different relative levels of RNA. © 1988 Academic Press, Inc.

Interleukin-1 (IL-I) , originally characterised as an

endogenous pyrogen and lymphocyte activation factor is now

known to mediate a wide range of biological activities and

attention has focused on the roles of IL-I in the pathogenesis

of diseases such as rheumatoid arthritis. IL-I increases

production of prostaglandins, collagenase, acute phase

proteins, accelerates cartilage degredation, and bone

resorption (reviewed by Dinarello, I). IL-I has been shown to

regulate gene expression in diverse cell types such as

Abbreviations used: IL = interleukin; TNF = tumour necrosis factor; GMCSF = Granulocyte Macrophage- Colony Stimulating Factor; PDGF = Platelet Derived Growth Factor; LT = lymphotoxin; LPS = lipopolysaccharide; PMA = phorbol 12- myristate 13-acetate; CHX = cycloheximide; PBM = peripheral blood mononuclear cells.

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 830

Vol. 156, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lymphocytes and adipocytes (I, 2). IL-I plays a role in both T

and B cell activation both directly (I), and possibly through

induction of other cytokines such as IL-6 (3).

There are two forms of IL-I which have been characterised and

molecularly cloned. These are designated as IL-I ~, the acidic

form (pI 5) and IL-18, the basic form (pI 7) (4). IL-I ~ and

IL-18 share significant structural homology and interact with

the same cell surface receptor (5). At present there is no

clear evidence for a differential function of these two IL-I

species, although it is known that the IL-I~ precursor is

biologically active, whereas the precursor IL-18 moiety has no

bioactivity (6). Analysis of IL-I species produced by different

cell types suggests the existence of tissue specific mechanisms

regulating IL-I production, as different cells contain

different relative amounts of IL-I~ and IL-I 8 which may vary

over 500 fold. Thus monocytes produce chiefly IL-18 (I, 4, 7),

while keratinocytes produce predominantly IL-I~ (8).

THP-I has previously been used in our laboratory to study

the regulation of MHC class II and IL-I regulation by cytokines

and its responses appear very similar to human monocytes (9).

It has been shown that THP-I produces two species of IL-I which

are biochemically indistinguishable from normal monocyte IL-I~

and IL-IH (10). Thus THP-I may represent a good model to study

IL-I gene regulation. We report here a major mechanism

controlling the differences in steady state levels of IL-I~ and

IL-I H mRNA is at the level of mRNA stability.

MATERIALS..AND. METHOD~

~ells and reagents THP-I cells were isolated from a girl with acute monocytic leukemia (11) and were a generous gift of Dr Kouji Matsushima NIH (Fredrick, ML). These were maintained and stimulated in RPMI 1640, supplemented with 25 mM HEPES, 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 U/ml streptomycin, (Gibco, Paisley, Scotland). THP-I was regularly tested for

831


mycoplasma infection and found to be negative. Salmonella Typhimurium LPS, phorbol myristate acetate (PMA), ~-amanitin and cycloheximide (CHX) were from Sigma (Dorset, England).

cDNA probes and RNA blot hybridisation The probe for IL-I~ (12) was a 460bp Eco RI-Bam HI insert corresponding to amino acids 1-132 of the IL-I precursor. The IL-IH probe (7) was a 530bp Nde I-Bam HI fragment corresponding to amino acids 1-139 of the IL-I ~ precursor Both were gifts of Dr. P.Lomedico (Hoffman La Roche, Nutley, New Jersey). The cDNA probe for 7B6 mRNA (a constitutively expressed mRNA) was a 708bp Pst I-Dra I fragment containing the Pst I- Dra I region from pBR322, this was the gift of Prof. U. Torelli (University of Modena, Italy). cDNA probes were labelled by random oligo priming (13) to 7-12 x 108 cpm/pg DNA using [~32p] dCTP (Amersham). Total cellular RNA was extracted by guanidinium isothiocyanate lysis as described previously (12) and slot blots were prepared by denaturing RNA in 15 x SCC, (20 x SSC = 3M NaCI, 300mM tri-sodium citrate, pH 7.0) 6% formaldehyde and applying~ RNA in 2-fold dilutions to a nitrocellulose filter using a Schleicher and Schuell minifold II. The filters were baked for 2 hours at 80°C and were prehybridised, hybridised, washed and autoradiographed as previously described (14).

Nuclear run off assay Cells were washed in Reticulocyte Standard Buffer (RSB =10mM Tris-HCl pH 7.4, 10 mM Na CI, 5mM Mg C12 , ImH dithiothreitol) and lysed by addition of Nonidet P-40 to a final concentration 0.2% (v/v). Nuclei were recovered by centrifugation through a 2 M sucrose cushion in a Sorvall HB-4 rotor at 10.O00 rpm for 15 min, and washed in RSB. 5 x 107 nuclei were stored in 100 pl of 50mM Tris HCI (pH 8.0), 5mM MgCI2, 0.1 mM EGTA, 40% (v/v) glycerol and I mM DTT at -70°C for up to I month.

For elongation of nascent transcripts 100 pl nuclei were incubated with 30pl 5 x Transcription buffer (25 mM Tris-HCl (pH 8.0) 12.5 mM MgCI2 750 mM KCI, 1.25 mM each of ATP, UTP and CTP) and 200 ~Ci (~32p)-GTP (3000 MCi/mmol, Amersham) for 30 mins at 30°C. In preliminary experiments incorporation of label was linear with respect to time. The reaction was terminated by addition of 20 U "RQI" DNase I (Promega Biotech) and 40wg transfer RNA. After 15 mins at 30°C the solution was made 150mM NaCI, 12.5 mM EDTA, I% SDS and proteinase K was added to a final concentration of 125 pg/ml and incubated for a further 20 min at 37°C. The mixture was extracted once with phenol:chloroform (2:1 vol/vol) and the organic phase was back extracted with an equal volume of 150mM NaCI, 12.5 mM EDTA, I% SDS, aqueous fractions were pooled and extracted with an equal volume of chloroform. The supernatant was applied to a I ml spun column of Sephadex G50 and the eluate ethanol precipitated.

Plasmid DNA was linearised with a restriction enzyme and 10 pg of each plasmid was denatured for 15 minutes in O.3M NaOH at 900C, quickly chilled on ice, and an equal volume of 2M ammonium acetate was added. The mixture was then filtered through nitrocellulose using a slot blot manifold. Single strand RNA probes were generated by in vitro transcription using SP6 polymerase (Promega biotech) and applied to filters in the same way as cellular RNA.

Filters were prehybridised in 50% Formamide, 4 x SSC, I% SDS, 2.5 x Denhardt's, 500pg/ml salmon sperm DNA, 200 pg/ml poly-(A) RNA and equal cpm of labelled RNA was hybridised to filters for 4 days at 42°C. Filters were washed in 2 x SSC O.1% SDS at room temperature, 2 x SSC containing 10 pg/ml RNase

832


also only the combination of both anti IL-le and IL-I~ antisera

reduced the counts to background (PHA only) response (Fig.l).

The kinetics of production and relative abundances of IL-I~ and

IL-18 mRNA were assessed by slot blotting. IL-IH mRNA was

detectable I hour after stimulation, levels increased

dramatically between I and 2 hours and continued to increase up

to 48 hours (Fig. 2). Extensive cell death occured after 48

hours thus, kinetics

followed furthur. The

similar to that of

of IL-18 mRNA expression could not be

accumulation of IL-I~ mRNA appeared

IL-18 except that IL-I~ mRNA was less

abundant (at 8 hours the apparrent decrease in IL-I~ mRNA is

due to less total RNA being present, compare 7B6 levels).

IL-I~ and IL-18 qenes are transcribed at a similar rate: We

next measured the rates of IL-I gene transcription in resting

and stimulated THP-I cells using the nuclear transcription

assay (Fig. 3). A low level of transcription of IL-I ~ and IL-I

7, __-- I i~

0 1 2

HOURS 4 0 2 6 8 PBM 8 IL -1Q i ~ :~:~:~ii~:

24 IL-18 ~ ' ~ O O I iii6i~il Q Q ~ !!iiiili2ili

Fiqure 2. kinetics of IL-I RNA accumulation: THP-I cells were stimulated with 10~g/ml LPS and 50ng/ml PMA and total cellular RNA extracted. RNA was applied to a nitrocellulose filter using a slot blot manifold. Duplicate filters were hybridised with 108 cpm/ml of each probe and after washing exposed to preflashed x-ray film for an equal period of time.

Fiqure 3. transcription of IL-I genes in isolated nuclei: Nuclei were isolated from THP-I cells after stimulation for different periods of time with PMA (50 ng/ml) and LPS (10 ~g/ml). Peripheral blood mononuclear cells were stimulated for 8 hours with PHA (1~g/ml) and PMA (50 ng/ml). Nuclear RNA was labelled, purified and equal cpm hybridised to antisense RNA immobilised on nitrocellulose filters for 4 days as described in materials and methods. Filters were waslled and exposed to x-ray film.

834


A for 10 min at room temperature then 0.1 x SSC 0.1% SDS 68°C for 10 min. Filters were exposed to film for 4 days.

Assay for IL-I IL-1 levels were measured using the thymocyte costimulator assay (15). Briefly, samples to be tested were mixed with neutralising antisera specific for IL-la or IL-18 and serially diluted and added to each well to give a final volume of 0.2 ml. Thymocytes from CBA mice were cultured at 106 cells/well in triplicate and were pulsed with IpCi 3H thymidine (90 Ci/mmol, Amersham) for the last 16 hours of a 72 hour incubation at 37°C in 5% CO 2 . Samples were harvested and tritiated thymidine incorporation was measured by liquid scintillation spectrophotometry.

RESULTS

THP-! produces predominantly IL-IH: Treatment of THP-I with

lipopolysaccharide (LPS) and phorbol myristate acetate (PMA)

induces rapid (within I hour) differentiation (11, data not

shown). Concomitant with this was the accumulation of

biologically active IL-I in the culture medium. The predominant

IL-I species was IL-I@ as nearly all of the activity was

neutralised by IL-18 specific polyclonal antiserum (Fig. I).

IL-I~ was also secreted by THP-I, since IL-I~ specific antisera

reduced the biological activity of the THP-I supernatant, and

1 2 0 "

1 0 0 "

x 8 0 "

~ 60" =

~ 40" N T 20' ...i

• , • , • , .

10 20 30 40 50

HOURS AFTERSTIMULATION

Fiqure I. kinetics of cytokine production: THP-I cells were stimulated with LPS (10pg/ml) and phorbol ester (PMA) (5Ong/ml) and supernatants were collected at various intervals thereafter and assayed for IL-I in the thymocyte costimulation assay using antisera specific for each form of IL-I. Mean counts per minute (cpm) ± standard error of the mean are shown; (D) conditioned medium only, (O) after incubation with sufficient anti IL- I~ to neutralise 100 units, (•) after incubation with sufficient anti IL-IB to neutralise 100 units, (O) incubation with both anti IL-I~ and IL-18. Response to PHA alone was 3774 ± 380 cpm.

833

V o l . 156 , No . 2, 1 9 8 8 B I O C H E M I C A L A N D B IOPHYSICAL RESEARCH C O M M U N I C A T I O N S

I I 133 -J d t,,.

z 1-, Z

I

0 +

0 ~ m

15 " " " ~ -- ' . ,

3 0 ~ ~ -.,-,

6 0 ~ " " ' ;~ - , -

120 --,,-

180 ~

15

3 0 ~ ~ - " .

6 0

120 ~ ~ - , . .

180 ~ ~ " - "

Fiqure 4. IL-I~ mRNA is less stable than IL-I~ mRNA: After stimulation for 2 hours with LPS (10 ~g/ml) and PMA (50ng/ml), 1.5 ~g/ml alpha amanitin was added to all of the cultures to inhibit RNA polymerase II, half of the cultures received 10 ug/ml cycloheximide and half did not. RNA was extracted at the indicated times and analysed by slot blotting as described in materials and methods. Autoradiographic exposures have been chosen so that the signal intensity for each probe at T=O is similar.

DISCUSSION

In this study we have examined the expression of II,-1~ and

IL-Ip genes by THP-I cells after treatment with the

combination of LPS and phorbol ester. The respective gene

products mediate very similar responses in a wide range of

biological assays, therefore it is of interest that each gene

appears to be regulated by distinct mechanisms. Several reports

(17, 18, 19) have characterised IL-Ip mRNA expression in

monocytic tumour lines. However none of these reports

described IL-I~ expression. Consistent with findings on

isolated macrophages and blood mononuclear cells (4, 15) THP-I

expresses greater amounts of mRNA for IL-IH than for IL-I~ and

this is also evident at the level of secreted protein where

antisera specific for IL-1H neutralise greater than 90% of the

thymocyte costimulator activity (Fig.1 ). While the levels of

each mRNA reflect the levels of secreted protein, the kinetics

836


was detectable in unstimulated THP-I (Fig. 3), which is

consistent with the low levels of IL-I mRNA which can be

detected on long exposures of RNA blots (data not shown). Upon

stimulation of THP-I, transcription rates of both IL-I~ and

IL-I~ genes was elevated at all time points tested (2, 6, and 8

hours). It was of interest to discover that the relative rates

of transcription for IL-I~ and IL-18 appeared similar in THP-I,

while in peripheral blood mononuclear cells (PBM) IL-IH was

transcribed at a much greater rate than IL-I~ (Fig. 3). This

suggests that in PBM differential rates of transcription

contribute to the higher relative levels of IL-18 mRNA which

have been reported (4, 15), while in THP-I cells transcription

is not the major mechanism determining the higher steady state

levels of IL-18 mRNA.

IL-I~ and IL-I@ mRNA have different stabilities: The different

steady state levels of IL-I~ and IL-18 mRNA appear to be

regulated post-transcriptionally in THP-I. This could include

differences in processing of nuclear precursors, transport

from nucleus to cytoplasm or intrinsic stability of mRNA. We

measured the half life of IL-I~ and 8 mRNA species after

blocking transcription with ~-amanitin, a specific inhibitor of

RNA polymerase II (16). The half life of IL-I~ mRNA was

estimated to be 30-60 minutes, while IL-18 mRNA did not go

through a half life during the 180 minute test period (Fig.

4). The half life of 7B6 mRNA was also longer than that of

IL-I~ suggesting that the rapid decay of IL-I~ mRNA is not due

to cell death or other non specific degredative mechanisms.

When cycloheximide (CHX) was added to the cultures there was a

marked stabilisation of IL-I~ mRNA suggesting a role for

protein synthesis in the regulation of IL-I~ mRNA decay, no

effect on IL-IH mRNA was observed in the presence of CHX.

835


of mRNA accumulation differ from the kinetics of protein

secretion (compare Fig. I and Fig. 2). This is most evident at

later time poiDts where the amount of protein detected has

plateaued while mRNA continues to accumulate. This may reflect

a mechanism of post-transcriptional control first described by

Knudsen and colleagues (20) in which prostaglandins which are

induced by LPS and PM~, block translation of IL-I mRNA but do

not influence mRNA levels. The rate of transcription of IL-I~

and IL-I~ does not reflect the steady state mRNA levels as both

genes are transcribed with equal efficiency (Fig. 3). This does

not appear to be an artefact of the transcription assay as

peripheral blood mononulear cells which contain primarily

lymphocytes and some monocytes and large granular lymphocytes

(all of which may produce IL-I) transcribe primarily IL-I~. In

addition tile experiment was repeated three times using either

single strand specific RNA probes or double stranded cDNA

probes. From these studies a post-transcriptional mechanism

appears to be the major determinant of the relative steady

state levels of IL-le and IL-I~. In support of this we show

that IL-I~ mRNA is unstable ( T I/2 = 30-60 minutes) while that

of IL-18 is stable (T I/2 > 180 minutes). The use of e-amanitin

to inhibit transcription by RNA polymerase II excludes

potential artefacts caused by agents such as actinomycin D

which have the potential to bind to RNA and alter its

metabolism. The protein synthesis inhibitor CHX prevented the

rapid decay of IL-I~ mRNA (Fig. 4). IL-le is very similar in

this respect to other cytokine mRNAs, such a Granulocyte-

Macrophage colony stimulating factor ( 21 ), Tumor Necrosis

Factor, Lymphotoxin, IL-6 (22), and Platelet Derived Growth

Factor (PDGF) B chain (23). The regulation of IL-18 appears to

be distinct from these other cytokines and more similar to

PDGF-A chain which was shown to have a longer half life than

837


PDGF-B chain in a fibroblast cell line (23). It remains to be

determined whether the differential levels of IL-I mRNA

expressed by other cells such as keratinocytes, where IL-I~

mRNA is the predominant species (8) are also regulated by post-

transcriptional mechanisms. It is tempting to speculate that a

family of tissue specific factors exist which confer

differential stability of cytokine mRNA possibly through

interaction with a common nucleotide sequence located in the 3'

untranslated region (21) , This is analogous to the well

documented tissue specific regulators of transcription (24).

Such a concept should be experimentally verifiable.

ACKNOWLEDGMENTS: We thank Dr. M. Shepard (Genentech, San Francisco) ,Dr G. Adolf (Boeringer Ingelheim, Vienna), Drs. P. Lomedico and A. Stern (Hoffman la Roche, Nutley, New Jersey), Dr.U. Torelli (Modena, Italy) for reagents used in this study. We thank all our colleagues who made useful suggestions. This work was funded by the Nuffield foundation (Oliver Bird grant), the Arthritis and Rheumatism Council and the Sunley trust.

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post-transcriptional control of il-1 gene expression in the acute monocytic leukemia line thp-1

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