Toxicology Letters, 53 (1990) 327-331 Elsevier 321 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 1990) (Revision received 25 May 1990) (Accepted 28 May 1990) Key words; Immunotoxicity; In vitro; mRNA; Cytokines; Macrophages SUMMARY The immunomodulatory agent Biostim (RU-41740) was investigated for its ability to induce the expres- sion of cytokine mRNAs in murine peritoneal macrophages in vitro. Northern blot analysis showed that in quiescent macrophage populations, both IL-la and IL-la mRNA levels were dramatically increased in response to 1 ,ug/ml Biostim. Dot-blot analysis showed that in quiescent macrophage populations the expression of mRNAs for IL-la, IL-IB, IL-6 and TNF-G( could be elevated by concentrations of Biostim as low as l-10 pg/ml, detectable after 3 h exposure. In parallel experiments LPS was effective only at the higher concentration of 10 ng/ml. Time-course analysis showed that the expression of these cytokine mRNAs was transient, peaking after l-3 h; only transcripts of IL-IS were detectable after 23 h exposure. No effects were seen on the expression of actin, a high-turnover housekeeping gene. We propose that this type of analysis represents a sensitive, specific and reproducible method for assessing the ability of drugs and chemicals to modulate the expression of cytokines that play a pivotal role in the induction of the immune response. Address for correspondence: Clive Meredith, Immunotoxicology Department, British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SMS 4DS, U.K. Abbreviations: IL-l = interleukin 1; IL-6 = interleukin 6; TNF = tumour necrosis factor; LPS = lipopo- lysaccharide; BSA = bovine serum albumin; SDS = sodium dodecyl sulphate. 0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Transcript
Toxicology Letters, 53 (1990) 327-331
Elsevier
321
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 1990)
(Revision received 25 May 1990)
(Accepted 28 May 1990)
Key words; Immunotoxicity; In vitro; mRNA; Cytokines; Macrophages
SUMMARY
The immunomodulatory agent Biostim (RU-41740) was investigated for its ability to induce the expres-
sion of cytokine mRNAs in murine peritoneal macrophages in vitro. Northern blot analysis showed that
in quiescent macrophage populations, both IL-la and IL-la mRNA levels were dramatically increased
in response to 1 ,ug/ml Biostim. Dot-blot analysis showed that in quiescent macrophage populations the
expression of mRNAs for IL-la, IL-IB, IL-6 and TNF-G( could be elevated by concentrations of Biostim
as low as l-10 pg/ml, detectable after 3 h exposure. In parallel experiments LPS was effective only at the
higher concentration of 10 ng/ml. Time-course analysis showed that the expression of these cytokine
mRNAs was transient, peaking after l-3 h; only transcripts of IL-IS were detectable after 23 h exposure.
No effects were seen on the expression of actin, a high-turnover housekeeping gene. We propose that this
type of analysis represents a sensitive, specific and reproducible method for assessing the ability of drugs
and chemicals to modulate the expression of cytokines that play a pivotal role in the induction of the
immune response.
Address for correspondence: Clive Meredith, Immunotoxicology Department, British Industrial Biological
Research Association, Woodmansterne Road, Carshalton, Surrey SMS 4DS, U.K.
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 cy-
totoxicity assay for TNF [17]. The overall findings ably demonstrate that the mono-
cyte/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 cul-
tured murine peritoneal macrophages as a sensitive specific indicator of the immuno-
modulatory potential of drugs and chemicals. In our initial studies we chose the com-
pound Biostim along with bacterial lipopolysaccaride (LPS) as model immuno-
stimulatory compounds to characterise our test system. Our results were initially con-
founded by findings that cultured peritoneal macrophages produce a pulse of cyto-
kine mRNA synthesis, including IL-l, IL-6 and TNF, simply as a result of the adher-
ence 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 cyto-
kine mRNA synthesis by overnight (16 h) incubation at 37°C. Subsequent adminis-
tration 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 time-
course of expression of mRNA for immunoregulatory cytokines in response to im-
munomodulatory 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 solu-
tion 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 manipu-
lation were prepared in double-distilled water previously treated with diethyl pyro-
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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 dur- ing 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 atmos- phere 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-pos- itive granules compared to non-elicited peritoneal cells. Macrophages were then cul- tured 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 cul- tures which were then further incubated for periods of up to 24 h. At defined time- points macrophage populations were gently washed in their own supernatant and ad- herent cells were lysed in the presence of 7.6 M guanidine hydrochloride using a mo- dified 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 incu- bated 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% forma- mide, 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 max- imised by baking at 80°C for 2 h.
For dot-blot analysis, total RNA (derived from 2 x lo6 macrophages) was dis- solved in a buffer containing 8.4% formaldehyde, 6.67 x SSC (standard saline ci- trate) (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-hybrid-
isation buffer [50% formamide, 5 x Denhardt’s (Denhardt’s = 0.02% polyvinylpyr- rolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 0.1% SDS, 150 pug/ml denatured 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% forma- mide, 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 radiola- belled cDNA probes (IL- 1 a, IL- l/3, IL-6, TNF-u and actin) were prepared at a specif- ic 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 per- formed 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 quanti- fied 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 pre- dicted sizes [2 1,221 and that (b) the RNA prepared was sufficiently clean after a single ethanolic precipitation to justify the use of the non-size fractionating technique, dot- blotting, in subsequent experiments to facilitate multiple sample processing.
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2.4
0.24
Kb
actin IL-lcr IL- lJ3
+ = Biostim lug/ml
Fig. 1. Northern blot analysis of macrophage mRNAs following exposure to Biostim. Quiescent macro-
phages were exposed to Biostim (1 pg/ml) for 3 h at 37°C; total RNA was isolated from macrophages,
fractionated on agarose gels and Northern-blotted. Blots were hybridised with 3ZP-cDNA probes for IL-
la, IL-lb and actin, stringently washed and exposed to autoradiographs at -70°C for up to 7 days. RNA
molecular weight markers 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) con-
In order to demonstrate that the effect of Biostim on these macrophage popula-
tions 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 concentra-
tions. Although systemic administration of BSA is known to evoke an immune re-
sponse when administered systemically in vivo, it has not been shown to induce cy-
tokine 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 popula-
tions 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 con-
centrations 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 immunomodu-
latory activity; Figure 3 shows that LPS was capable of eliciting a similar spectrum
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Time 0 1 2 3 4 6 8 23 (hrl
IL-6
TNF(r
actin
Fig. 4. Dot-blot analysis of the time-course of expression of macrophage mRNAs following exposure to
Biostim. Quiescent peritoneal macrophages were exposed to Biostim (I fig/ml) for various times as indicat-
ed in the figure before cell disruption and isolation of RNA. Descending rows represent sequential 5-fold
dilutions 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 steady- state 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
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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 sensitive to Biostim and elevations in cytokine mRNA
levels were detectable using concentrations on Biostim as low as 10 pg/ml. Sensitivity
of the assay was considerably enhanced by allowing the adherent macrophage popu-
lations 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 pro-
duced by distinct cell populations [24]. Experiments using in situ hybridisation analy-
sis will be able to address this possibility directly.
Our experiments suggest that the activity of Biostim in elevating cytokine gene ex-
pression 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 cy-
tokine transcription at very low concentrations. Experiments using purified compo-
nents 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 stud-
ied. Recently it has been demonstrated that Biostim can induce the release of a tu-
mour-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 detect-
ing 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 them-
selves 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 differen- tiate 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 expres- sion 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 possi- ble, at present, to provide only semi-quantitative results using this procedure, recent developments suggest that absolute quantitation can be provided by including cyto- kine 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 im- munomodulatory 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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