The effect of Biostim (RU-41740) on the expression of cytokine mRNAs in murine peritoneal macrophages in vitro

321

Toxicology Letters, 53 (1990) 327-331 Elsevier

TOXLET

02449

The effect of Biostim (RU-41740) on the expression of cytokine mRNAs in murine peritoneal macrophages in vitro

Clive Meredith’, Mary P. Scott’, Hadewijch Pekelharing2 and Klara Miller1 Immunotoxicology Department, British Industrial Biological Research Association, Carshalton, Surrey (U.K.) and zDepartment of Toxicology, Agricultural University, Wageningen (The Netherlands) (Received

21 February

(Revision

received 25 May 1990)

1990)

(Accepted

28 May 1990)

Key words; Immunotoxicity;

In vitro; mRNA;

Cytokines;

Macrophages

SUMMARY The immunomodulatory sion of cytokine in quiescent in response expression

agent Biostim (RU-41740)

mRNAs

in murine

macrophage

populations,

to 1 ,ug/ml Biostim. of mRNAs

for IL-la,

as low as l-10 pg/ml, detectable higher mRNAs

concentration

peritoneal

peaking

and IL-la

analysis

showed

for its ability to induce the expres-

in vitro. Northern mRNA

that in quiescent

macrophage

In parallel

analysis

experiments

showed

after l-3 h; only transcripts

that

the expression

of IL-IS were detectable

of actin, a high-turnover

housekeeping method

immune

a sensitive,

the expression

the

of cytokines

of these cytokine after 23 h exposure.

gene. We propose

for assessing

that play a pivotal

of Biostim

that this

the ability

of drugs

role in the induction

of the

response.

Address for correspondence: Clive Meredith, Research

populations

LPS was effective only at the

Time-course

specific and reproducible

to modulate

that

increased

after 3 h exposure.

type of analysis and chemicals

showed

by concentrations

No effects were seen on the expression represents

blot analysis

levels were dramatically

IL-IB, IL-6 and TNF-G( could be elevated

of 10 ng/ml.

was transient,

both IL-la

Dot-blot

was investigated

macrophages

Association,

Abbreviations: IL-l lysaccharide;

Woodmansterne = interleukin

1; IL-6 = interleukin

BSA = bovine serum albumin;

0378-4274/90/$3.50

Immunotoxicology

Road, Carshalton,

Department,

6; TNF

= tumour

SDS = sodium dodecyl

@ 1990 Elsevier Science Publishers

British Industrial

Biological

Surrey SMS 4DS, U.K. necrosis

sulphate.

B.V. (Biomedical

Division)

factor;

LPS = lipopo-

328

INTRODUCTION

Biostim (RU-41740)

is an extract from Klebsiellu pneumoniae which is substantially

composed of two high-molecular-weight glycoproteins 350 kDA and 95 kDA. The approximate composition of the extract is 68% carbohydrate, 24% protein, 2% nucleic acid and 3% lipid [ I] and it has been shown to possess immunomodulatory activity both in animals [2-61 and in humans [1,7-IO]. Both lymphocyte activities [l,l l-131 and monocyte activities [14,10,15] are demonstrably affected. Previous studies [ 151 have demonstrated that nanogram concentrations of Biostim are able to increase the production of IL-l and TNF in human monocytes cultures in vitro; these assays were conducted using the familiar thymocyte co-stimulator assay for IL-l [16] and the cytotoxicity assay for TNF [17]. The overall findings ably demonstrate that the monocyte/macrophage is likely to be the primary target for the activity of Biostim. We have recently been investigating the analysis of cytokine mRNA levels in cultured murine peritoneal macrophages as a sensitive specific indicator of the immunomodulatory potential of drugs and chemicals. In our initial studies we chose the compound Biostim along with bacterial lipopolysaccaride (LPS) as model immunostimulatory compounds to characterise our test system. Our results were initially confounded by findings that cultured peritoneal macrophages produce a pulse of cytokine mRNA synthesis, including IL-l, IL-6 and TNF, simply as a result of the adherence procedure used to purify peritoneal macrophage populations in vitro [Meredith, Scott and Miller, manuscript in preparation]. We therefore elected to allow our isolated macrophage populations to adhere to tissue-culture-ware for 1 h before washing and then allowed them to become essentially quiescent with respect to cytokine mRNA synthesis by overnight (16 h) incubation at 37°C. Subsequent administration of test compound permitted the analysis of subtle changes in the steady-state levels of cytokine mRNA and permitted us to determine the dose-response and timecourse of expression of mRNA for immunoregulatory cytokines in response to immunomodulatory agents. We report here that concentrations of Biostim as low as l-l 0 pg/ml dramatically elevate the levels of expression of mRNAs for IL- 1~1,IL- lfi, TNF-a and IL-6 and that the effects were transient with steady-state levels of mRNA peaking

at 2-3 h post-exposure.

MATERIALS

AND METHODS

Biostim (RU-41740) was supplied by Dr. A. Vecchi, Mario Negri Institute, Milan and was dissolved in RPM1 1640 medium with gentle mixing for 30 min. Stock solution was prepared at 100 x the final concentration required for the in vitro exposure and was prepared fresh for each experiment. Bacterial lipopolysaccharide (LPS) was from Sigma (from E. coli 055:B5) and was prepared as for Biostim. All chemicals employed were of Analar or equivalent grade. Solutions employed for RNA manipulation were prepared in double-distilled water previously treated with diethyl pyro-

329

carbonate (0.1%) and then autoclaved. Glassware was rendered ribonuclease-free by baking at 250°C for 3 h; normal precautions were taken to avoid contamination during RNA manipulations. Culture preparation

Female C3H mice of age 4-6 weeks (Charles River) were injected intraperitoneally with an eliciting agent of 1.5% thioglycolate broth (sterilised and aged in the dark for 2 months); 4 days later, animals were killed by CO2 inhalation and macrophages harvested from the peritoneal cavity by repeated irrigation with normal saline at 37°C. Cells were recovered by centrifugation at 1000 x g for 15 min and resuspended in RPM1 1640 medium (Gibco) at a concentration of lo6 cells/ml. Cell suspensions (2 ml) were incubated in plastic dishes (Nunc, 35 mm) for 1 h at 37°C in an atmosphere of 5% CO2 in air and adherent macrophages were purified by repeated washing. This population consisted primarily of ‘mature’ macrophages containing many cells showing long cytoplasmic processes and increased numbers of acid-phosphatase-positive granules compared to non-elicited peritoneal cells. Macrophages were then cultured for 1620 h at 37°C in RPM1 1640 medium containing 10% foetal calf serum (Flow Laboratories) and 62.5 IU/ml-63.5 pg/ml of penicillin-streptomycin to enable the cells to become quiescent with respect to the expression of immunoregulatory genes. Treatment

of cell cultures

Stock solutions of test compounds (Biostim, LPS, bovine serum albumin (BSA) (Sigma) or media control) were diluted lOO-fold into the quiescent macrophage cultures which were then further incubated for periods of up to 24 h. At defined timepoints macrophage populations were gently washed in their own supernatant and adherent cells were lysed in the presence of 7.6 M guanidine hydrochloride using a modified procedure previously described [18]. Briefly, 0.5 ml of guanidine buffer (7.6 M guanidine hydrochloride, 0.1 M potassium acetate, 10 mM dithiothreitol, pH 5.0) was added to the adherent macrophage layer on the plates, swirled gently and incubated for 5 min. The plates were rinsed with a further 0.5 ml of guanidine buffer which was pooled with the original lysate. DNA in the lysate was sheared by repeated passage through a 25 g needle and 0.6 vol. of 95% ethanol was added with vigorous mixing. Following overnight storage at -20°C the specifically precipitated RNA was recovered by centrifugation at 13 000 x g for 20 min. The pellet was washed in 90% ethanol and dried in vacua for 10 min. Northern

and dot blotting

For Northern blot analysis, total RNA was dissolved in sample buffer [57% formamide, 7.6% formaldehyde, 0.04 M morpholinopropanesulfonic acid, 10 mM sodium acetate, 1 mM ethylenediaminetetraacetic acid (EDTA)] by incubating at 70°C for 10 min and aliquots (approx. 5 pg of total RNA) were fractionated on 1.5% agarose

330

gels under denaturing conditions as described previously [19]. Fractionated RNA was Northern blotted to a Nytran membrane (Schleicher and Schuell) essentially as described by the supplier; covalent binding of macromolecule to membrane was maximised by baking at 80°C for 2 h. For dot-blot analysis, total RNA (derived from 2 x lo6 macrophages) was dissolved in a buffer containing 8.4% formaldehyde, 6.67 x SSC (standard saline citrate) (SSC = 150 mM sodium chloride, 15 mM trisodium citrate), by incubation at 65°C for 20 min. Aliquots (100 ~1) were dot-blotted onto Nytran membranes using a 96-well vacuum manifold (Bethesda Research Laboratories) and macromolecules covalently bound by baking as described above. Hybridisation analysis

Nytran membranes from Northern or dot-blotting were incubated in a pre-hybridisation buffer [50% formamide, 5 x Denhardt’s (Denhardt’s = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.1% SDS, 150 pug/mldenatured salmon sperm DNA, 5 x SSPE (standard saline phosphate-EDTA) (SSPE = 180 mM sodium chloride, 10 mM sodium phosphate, 1 mM EDTA, pH 7.41 for 24 h at 42°C. The membranes were then transferred to a hybridisation buffer (50% formamide, 1 x Denhardt’s, 150 pg/ml denatured salmon sperm DNA, 0.1% SDS, 5 x SSC) containing the appropriate 32P-cDNA probe at lo6 c.p.m./ml. These radiolabelled cDNA probes (IL- 1a, IL- l/3, IL-6, TNF-u and actin) were prepared at a specific activity of 5 x lo*-lo9 c.p.m./pg using the random priming reaction essentially as described previously [20]. Membranes were hybridised for up to 16 h at 42°C and were then stringently washed using the supplier’s protocol. The final wash was performed in the presence of 1% SDS, 0.1 x SSC at 56°C. The membranes were then exposed to X-ray film (Fuji) for 2-7 days at -70°C. Autoradiographs were quantified by scanning video densitometry. RESULTS

Initially, we investigated the capacity of Biostim to influence the expression of mRNAs for IL-la and IL-l/? in murine peritoneal macrophage populations which had been cultured overnight to minimise interference from the pulse of cytokine mRNA synthesis which accompanies adherence of the macrophage to culture-ware. Northern blot analysis (Fig. 1) clearly showed that the administration of Biostim (1 pg/ml) to these quiescent macrophage cultures had no effect on the steady-state levels of mRNA transcripts for actin, but dramatically elevated the levels of mRNA for both IL-la and IL-lp, analysed after 3 h exposure. This Northern blot analysis also demonstrated that (a) the mRNA extracted from the lysates was of high integrity since the detected bands of both IL-la and IL-1B mRNA corresponded to the predicted sizes [2 1,221and that (b) the RNA prepared was sufficiently clean after a single ethanolic precipitation to justify the use of the non-size fractionating technique, dotblotting, in subsequent experiments to facilitate multiple sample processing.

331

2.4

0.24 Kb IL-lcr

actin

+ = Biostim Fig. 1. Northern phages

blot analysis

were exposed

fractionated la, IL-lb molecular

on agarose

(1 pg/ml)

mRNAs

washed

following

exposure

to Biostim.

for 3 h at 37°C; total RNA was isolated

gels and Northern-blotted.

and actin, stringently weight markers

lug/ml

of macrophage

to Biostim

IL- lJ3

Blots were hybridised

and exposed to autoradiographs

Quiescent

with 3ZP-cDNA probes at -70°C

macro-

from macrophages, for IL-

for up to 7 days. RNA

are shown on the right.

Biostim

IL-la

IL-q3

Fig. 2. Dot-blot analysis of macrophage mRNA following exposure to Biostim or BSA. Quiescent murine peritoneal macrophages were exposed for 3 h at 37°C to the following concentrations of Biostim: (I) control, (2) 1 ng/ml, (3) 10 ng/ml, (4) 100 ng/ml, (5) 1 ,ug/ml, (6) 10 pg/ml, (7) 100 pg/ml; or of BSA: (8) 1 ng/ml, (9) 100 ng/ml, (10) 1 pg/ml, (11) 100 pg/ml. Descending rows represent sequential 5-fold dilutions of the upper row.

332

LPS

Biostim

IL-6

TNFff

actin

Fig. 3. Dot-blot

analysis

of macrophage

charide

(LPS). Quiescent

Biostim

or LPS. (1) control,

ng/ml,

(8) 1 pg/ml,

macrophages

mRNA

(2) 1 pg/ml,

(9) 10 pg/ml,

following

were exposed (3) 10 pg/ml,

(10) 100 pg/ml.

exposure

to Biostim

or bacterial

for 3 h at 37°C to the following (4) 100 pg/ml,

Descending

(5) 1 ng/ml,

rows represent

lipopolysac-

concentrations

(6) 10 ng/ml,

sequential

of

(7) 100

5-fold dilutions

of the upper row.

In order to demonstrate that the effect of Biostim on these macrophage populations was specific, we treated cultures with Biostim at concentrations ranging from 1 ng/ml to 100 pug/ml or with bovine serum albumin (BSA) at equivalent concentrations. Although systemic administration of BSA is known to evoke an immune response when administered systemically in vivo, it has not been shown to induce cytokine expression during in vitro culture with immune cells. Figure 2 shows that in no case did BSA cause any elevations in the level of IL-l mRNA transcripts, whereas Biostim caused elevations of both forms of IL-l mRNA at all concentrations tested at 3 h post-dosing. In order to titrate fully this response and to assess the effect of Biostim on other cytokine mRNAs such as IL-6 and TNF-or, macrophage populations were treated with concentrations of Biostim in the range 1 pg/ml-100 pg/ml; Figure 3 shows that for each of the cytokine mRNAs studied, IL-ltx, IL-l/l, IL-6 and TNF-a, Biostim caused a dose-dependent elevation in the level of transcripts at concentrations between 1 and 100 pg/ml, the effect being maximal at concentrations of 100 pg/ml and above. No effect was observed on the expression of actin mRNA at any of the concentrations studied. We also studied the effects of LPS on macrophage populations since it could be suggested that the LPS present in Biostim might be responsible for its immunomodulatory activity; Figure 3 shows that LPS was capable of eliciting a similar spectrum

333

Time

0

1

2

3 4

6

8 23 (hrl

IL-6

TNF(r

actin

Fig. 4. Dot-blot

analysis

Biostim. Quiescent

of the time-course

peritoneal

macrophages

ed in the figure before cell disruption dilutions

of expression

of macrophage

were exposed to Biostim

and isolation

of RNA.

mRNAs

following

exposure

to

(I fig/ml) for various times as indicat-

Descending

rows represent

sequential

5-fold

of the upper row.

of cytokine mRNA synthesis to Biostim, but was effective only at the much higher concentration of 10 ng/ml. In order to characterise better this response, we further determined the time-course of expression for each of these cytokine mRNA species in macrophage cultures in response to a saturating dose of Biostim (1 pug/ml). kigure 4 shows that the steadystate levels of mRNA for IL-la peaked at l-2 h post-exposure, whereas IL-l/I and IL-6 and TNF-a levels peaked at 2-3 h post-exposure. A decline in the levels of all cytokine mRNAs was evident at 6 h post-exposure and only IL- l/3 mRNA transcripts were weakly detected after 23 h. No effect was seen on the expression of actin mRNA transcripts at any of the time-points studied. DISCUSSION

The results obtained in these experiments clearly demonstrate

that Biostim has

334

profound

effects on the expression

of cytokine

mRNAs

in murine

peritoneal

macro-

phage populations, being capable of elevating the steady-state levels of mRNA for each of the 4 cytokines studied (IL-ltx, IL-l/?, IL-6 and TNF-a). These macrophage populations

were exquisitely

levels were detectable

sensitive

to Biostim

using concentrations

and elevations

in cytokine

mRNA

on Biostim as low as 10 pg/ml. Sensitivity

of the assay was considerably enhanced by allowing the adherent macrophage populations to become quiescent with respect to the expression of cytokine mRNAs by overnight culture, a situation which reflects the state of mRNA expression in freshly isolated macrophages. Time-course analysis of the elevation of cytokine mRNA expression shows that the effect is transient, peaking at l-3 h for the cytokines studied with only transcripts for IL-ID mRNA detectable after 23 h exposure. This may either reflect the relative stabilities of the mRNA species or may be connected with the fact that the beta-form of IL-l is the predominant form released by murine peritoneal macrophages [23]. It has been shown, at least in human peripheral blood, that IL-1~ and IL-l/? are produced by distinct cell populations [24]. Experiments using in situ hybridisation analysis will be able to address this possibility directly. Our experiments suggest that the activity of Biostim in elevating cytokine gene expression in macrophages is not directly attributable to the LPS content of the Biostim preparation. Although Klebsiella extracts are known to possess significant quantities of LPS [25,26], the activity of Biostim in the assays described here is detectable at concentrations which are lOO-fold lower than equipotent concentrations of LPS preparations. Either the LPS content of Biostim does not contribute to its activity or else LPS acts synergistically with another unidentified component to activate cytokine transcription at very low concentrations. Experiments using purified components of Biostim should resolve these possibilities. Biostim has previously been shown to induce the secretion of IL-l and TNF-like activity in human monocyte populations [ 151. The studies described here extend and clarify these findings by demonstrating at a molecular level that both forms of IL-l are induced as well as IL-6 and TNF-cr in the murine macrophage populations studied. Recently it has been demonstrated that Biostim can induce the release of a tumour-inhibitory factor in human monocyte populations [27]. Whether this factor is related to the TNF expression described here remains to be clarified. Clearly this type of molecular-biological analysis offers significant advantages over existing procedures for determining immunomodulatory potential of drugs and chemicals. We have demonstrated here that the technique is highly sensitive in detecting the immunopotentiating effect of Biostim on macrophage populations at 10 pg/ ml whereas functional assays in human monocytes are l&100-fold less sensitive [ 151. Relatively small numbers of cells are required for this analysis, most of the dot-blots described here were produced from lo5 cells. Samples can be multiple-screened using a battery of cDNA probes to detect mRNA species of interest and the blots themselves can be stripped and rescreened on up to 5 separate occasions. Furthermore,

335

inclusion of determination of a housekeeping gene such as actin allows us to differentiate between those effects related to toxic or subtoxic effects of a compound and those entirely due to the immunomodulatory properties of the xenobiotic. Future developments in this field involve an expansion in the range of molecular probes available in order to discern subtle and selective effects on altered gene expression and the analysis of effects on other cell populations, e.g. on human monocytes and other monocyte/macrophage populations of diverse origin. Although it is possible, at present, to provide only semi-quantitative results using this procedure, recent developments suggest that absolute quantitation can be provided by including cytokine mRNA standards derived from cDNAs cloned into expression vectors [28]. We propose that this sensitive, specific, reproducible methodology is a valuable tool for the analysis of cytokine expression and therefore has applications in studying the immunomodulatory potential of drugs and industrial chemicals. ACKNOWLEDGEMENTS

We are grateful to the following for their gifts of murine cDNAs: P. LoMedico, Hoffmann-LaRoche (IL-la); P. Gray, Genentech (IL-l/Q W. Fiers, Biogen (TNF-a); C.-P. Chiu, DNAX (IL-6). This work was partially supported by CEC contract No. BAP-0272-UK to whom our thanks are due.

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