Altered cytokine (receptor) mRNA expression as a tool in immunotoxicology

Toxicology 130 (1998) 43–67

Review

Altered cytokine (receptor) mRNA expression as a tool inimmunotoxicology

Rob J. Vandebriel a,*, Henk Van Loveren a, Clive Meredith b

a Laboratory for Pathology and Immunobiology, National Institute of Public Health and the En6ironment,PO Box 1. 3720 BA Biltho6en, The Netherlands

b Immunotoxicology, BIBRA International, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK

Received 22 February 1998; accepted 16 June 1998

Abstract

Molecular immunotoxicology is aimed at analysing exposure effects on the temporal expression of importantimmunoregulatory genes. Cytokines play key roles in the immune system and thus molecular immunotoxicology hasfocused on the analysis of cytokine (expression) levels. These targets offer important new avenues to explore both interms of mechanistic understanding of immunotoxicity and in terms of developing new assays and tests for predictingthe immunotoxic potential of novel compounds. Effects on cytokine levels can be analysed on two different levels,these being mRNA and protein. The choice essentially depends on the aim of the study. Proteins comprise thebiological activity so they are a more direct measure than mRNA. mRNA on the other hand, measures at a specificpoint in time within a tissue or organ, whereas protein is measured in a body fluid, possibly as a spill-over from tissue,or in a supernatant as a summation over a culture period. mRNA levels are assayed using Northern or dot blottingthat both comprise hybridisation and using reverse transcription-polymerase chain reaction (RT-PCR). Although thelatter technique has both enormous sensitivity and relative ease of operation as important advantages, it requiresmuch more effort in terms of quantitation. References to the nucleic acid sequences of human, murine, and ratcytokines and their receptors are presented (with accession numbers). Examples in which molecular techniques weresuccessfully employed to assess immunotoxicity and (in some cases) understand mechanisms of action are alsopresented. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Immunotoxicology; Cytokine (receptor); Gene expression; Gene sequence; Hybridisation; RT-PCR

* Corresponding author. Tel.: +31-30-2742929; Fax: +31-30-2744437; E-mail: [email protected]

0300-483X/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0300 -483X(98 )00089 -4

R.J. Vandebriel et al. / Toxicology 130 (1998) 43–6744

1. Introduction

The advent of molecular biological techniquesinto the biological sciences has been revolutionaryboth in terms of basic biomedical sciences and ourunderstanding of disease processes and in terms ofapplied sciences and the development of the bio-technology industry. The discipline of toxicologyhas been relatively slow to appreciate the benefitsof this new technology and the opportunities thatit affords for understanding the mechanisms oftoxicity and for developing tests which may helpto predict the toxicity of novel compounds. How-ever, over the past 5 years, many toxicologistshave begun to acquire these new skills and toapply them to traditional areas of toxicologicalinvestigation, particularly in the fields of genetictoxicology and chemically induced carcinogenesis(Rumsby, 1993). The discipline of immunotoxicol-ogy, which is itself a relatively recent arrival, atleast on the regulatory scene, has begun to appre-ciate the potential benefits of a molecular biologi-cal approach to understanding immunedysregulation and is looking to the benefits whichsuch an approach may provide in terms of impor-tant early markers of immunotoxicity within invivo, ex vivo, and in vitro immunotoxicity studies.The purpose of this review is to provide anoverview of the technology available, to provideexamples of the successful exploitation of thetechnology in immunotoxicity testing and to buildupon basic concepts which were laid down in anearlier review (Meredith, 1992).

2. Rationale for molecular approaches toimmunotoxicology

2.1. Role of cytokines in immune regulation

The essence of a molecular approach to im-munotoxicology is the analysis of exposure effectson the temporal expression of important im-munoregulatory genes. Cytokines play key rolesin the ontogeny and maintenance of the immunesystem as well as the activation and differentiationof cells of this system. Importantly, also cells thatdo not belong to the immune system, such as

endothelial cells, produce cytokines. Furthermore,cytokines are able to link the innate immunesystem to the specific immune system. As a discus-sion on the role of the different cytokines andtheir receptors is beyond the scope of this review,we refer to review articles and references therein(Arai et al., 1990; Oppenheim et al., 1991; Durumand Oppenheim, 1993; Howard et al., 1993;Moore et al., 1993; Seder and Paul, 1994;Trinchieri, 1995; O’Garra, 1998). In addition, sev-eral handbooks (e.g. Nicola, 1994) list the differ-ent cytokines and their receptors with emphasison gene structure, regulation of expression, bio-chemical structure, signal transduction, cellularsources, cellular targets, and physiologicalfunctions.

2.2. Analysis of the modulation of cytokine(expression) le6els

From the key roles that cytokines play, molecu-lar immunotoxicology has focused on the analysisof cytokine (expression) levels within models ofimmune dysregulation. Besides, as ongoing im-mune responses can be monitored by assessingcytokine levels, these determinations have the po-tential to be early indicators of immunotoxicity.These targets offer important new avenues toexplore both in terms of mechanistic understand-ing of immunotoxicity and in terms of developingnew assays and tests for predicting the im-munotoxic potential of compounds.

2.3. Analysis of cytokine le6els 6s. cytokineexpression

Analysis of the synthesis and secretion ofpolypeptides and proteins, either by release intothe peripheral blood in vivo or by release into thecell culture supernatant in vitro is a well estab-lished technique which can be performed either bybioassay or by specific macromolecular detectione.g. ELISA. This type of assay forms the back-bone of many immune function tests. An impor-tant advantage of ELISA over analysis of mRNAlevels to be described below is that it is the proteinthat comprises the biological activity. In addition,measuring mRNA expression requires relatively

R.J. Vandebriel et al. / Toxicology 130 (1998) 43–67 45

laborious techniques, hampering its implementa-tion in immunotoxicity screening. An importantdrawback of ELISA over analysis of mRNAlevels is that cytokines can only be measured inbody fluids (blood, urine, peritoneal fluid, nasallavage, bronchoalveolar lavage) or cell superna-tants but not in (intact) tissue. Cytokines may,in some cases, exert their effects only within acertain organ or tissue so only a spill-over effectinto e.g. the peripheral blood is measured. Fur-thermore, the measurement of cytokine releaseinto cell culture supernatant is essentially a cu-mulative measure and unless time points arechosen very carefully, will not yield informationon the way in which cells are responding tovarious stimuli and how a cascade of events isprogressing. Generally the rate of production ofa cytokine in this type of assay is greater thanthe rate of degradation (by receptor binding andby non-specific proteolysis) and thus the mea-surement will be representative only for the sumcumulation of events. Since we know that im-mune responses involve a variety of cell typesand sequences of events, we may not alwaysidentify the crucial alterations in gene expressionand therefore crucial events responsible for theimmunotoxic manifestation. Thus, cytokine mea-surements in body fluids or cell supernatants donot always provide the proper or complete pic-ture. ELISA or bioassay can, however, still be apowerful tool in immunotoxicity testing. Themajority of mRNA species are generally short-lived, existing only to convey information fromnucleus to cytoplasm and thereafter beingrapidly degraded by endogenous ribonuclease.This instability is of great analytical advantagein that it allows us to pinpoint very preciselywhat cells are doing at a given moment in timeand how it is responding to various externalstimuli.

A further point of interest is the relationshipbetween altered mRNA expression and proteinlevels. It is clear that they may not always di-rectly be related, since cytokines can, e.g. bestored intracellularly. Systematic studies on thisissue are lacking. Regarding the sensitivity ofELISA compared to measuring mRNA expres-sion, reverse transcription-polymerase chain reac-

tion (RT-PCR) is especially able to detectminute quantities of mRNA, whereas in somecases ELISA is of insufficient sensitivity (Vande-briel et al., in preparation).

2.4. Information on cytokine gene sequences

A large number of cytokines and their recep-tors has been cloned at both cDNA and ge-nomic level thus fortuitously facilitating theexploitation of molecular biology within disci-plines such as immunotoxicology. cDNA clonescan be used to generate probes which are idealreagents with which to study gene expression atthe mRNA level and thus to assess modulationof this expression in response to various stimuli.Alternatively the sequence information madeavailable either in the literature or via databases (see below), can facilitate the design ofappropriate oligonucleotide probes or primersfor use in RT-PCR analysis. Although most ofthe early cDNA probes were for human ormouse (Table 1), more rat clones are becomingavailable. This is particularly fortunate since therat is the species most widely used in toxicologytesting although the mouse has a particularniche in certain types of immune function test-ing. In our analyses we have sometimes beenable to cross-hybridise murine or human probeswith rat mRNAs, e.g. we have successfully usedmurine IL-1 probes to detect rat mRNA sincethere is a 83% homology between the species(Nishida et al., 1989) whereas for IL-3 there ap-pears to be less homology and little prospect forcross-species hybridisation (Yang et al., 1986).As each cytokine is identified and sequenced, asearch begins for it’s specific receptor. A signifi-cant amount of information now exists on cy-tokine receptor gene sequences (Table 2). Thisprovides us with tools to understand im-munotoxicity through regulation at the tran-scriptional level of both cytokines and theirreceptors. We have included accession numbersto easily obtain these sequences from databaseson the Internet, such as from EMBL (http://www.ebi.ac.uk/queries/quiries.html) and NCBI(http://www.ncbi.nlm.nih.gov).


Table 1Availability of sequence data for cytokines from human, mouse and rat

MouseHumanCytokine/ Ratgrowth factor

X01450M15329IL-1a D00403Nishida et al., 1987 Lomedico et al., Nishida et al.,

19891984M15330IL-1b M98820M15131Nishida et al., 1987 Gray et al., 1986 Liu et al., 1995J00264 K02797/X01772IL-2 M22899

Kashima et al., McKnight et al.,Taniguchi et al., 1983; Devos et al., 1983; Maeda et al., 1983; Mita etal., 1983; Fujita et al., 1983; Clark et al., 1984 19891985

X03846K01850IL-3 M14743Yang et al., 1986 Fung et al., 1984 X03914

Cohen et al.,K032331986Miyatake et al.,

1985a

X16058M13238IL-4 M13982McKnight et al.,Lee et al., 1986Yokota et al., 19861991

X06271 X54419IL-5 X04688Campbell et al., Uberla et al.,Azuma et al., 1986

19911988M14584 J03783 M26744IL-6May et al., 1986 Northemann etChiu et al., 1988

al., 1989X07962IL-7 J04156

Goodwin et al., 1989 Namen et al.,1988

M28130IL-8Mukaida et al., 1989M30135 M30136 L36460IL-9

Flubacher et al.,Renauld et al.,Renauld et al., 19901990 1994

M57627 M37897IL-10 L02926Goodman et al.,Moore et al.,Vieira et al., 199119921990

IL-11 U03421M37006/M57765Paul et al., 1990 Morris et al.,

1996M86671M65290IL-12 p40

Wolf et al., 1991 Schoenhaut et Mathieson andal., 1992 Gillespie, 1996

M38444/M65272Gubler et al., 1991M65291 M86672IL-12 p35

Schoenhaut etWolf et al., 1991al., 1992

M38443/M65271Gubler et al., 1991


Table 1 (continued)

MouseHumanCytokine/ Ratgrowth factor

X69079 L13028IL-13 L26913Lakkis and Cruet,McKenzie et al.,Minty et al., 1993

1993b 1993L06801McKenzie et al., 1993aL13029McKenzie et al., 1993b

IL-14 Ambrus et al., 1996U14407 U14332IL-15Grabstein et al., 1994 Anderson et al., Reinecker et al.,X91233 19961995aKrause et al., 1996

AF006001IL-16 U82972Baier et al., 1997 Keane et al., 1998U32659IL-17 U43088Yao et al., 1995a Kennedy et al., Kennedy et al.,

19961996D49950IL-18 U77776D49949

Okamura et al., Conti et al., 1997Ushio et al., 19961995X02611TNF-a X01394

Shirai et al., 1989Fransen et al.,Pennica et al., 1984; Shirai et al., 19851985M10988

Wang et al., 1985 M11731Pennica et al., 1985

J00219 K00083IFN-g X02325X02326Gray and Goeddel,Taya et al., 1982; Gray et al., 1982; Devos et al., 1982; Gray and

1983Goeddel, 1982; Derynck et al., 1982, 1983 X02327Dijkema et al.,1985

X03438 M13926 U37101G-CSFHan et al., 1996Tsuchiya et al.,Nagata et al., 1986a

1986M13008Souza et al., 1986X03656Nagata et al., 1986bX05825 X05010M-CSF M84361

DeLamarter et al., Borycki et al.,Ladner et al., 198719931987

M21149M21952Ladner et al., 1988

X03021 X02333GM-CSFOaks et al., 1995Miyatake et al., 1985b Gough et al., 1985

X03020M10663Miyatake et al.,Wong et al., 19851985bM11220Stanley et al., 1985Lee et al., 1985

M13207Kaushansky et al., 1986

M13177 X52498TGF-b1 X02812Derynck et al., 1985 Derynck et al., Qian et al., 1990

1986


Table 2Availability of sequence data for cytokine receptors from human, mouse and rat

Cytokine/growth factor RatMouseHumanreceptor

M27492 M20658/M29572 M95578IL-1R type IHart et al., 1993Sims et al., 1988Sims et al., 1989Z22812X59769IL-1R type II X59770

McMahan et al., 1991 McMahan et al., 1991 Bristulf et al., 1994K02891 M55049K03122IL-2R a

Leonard et al., 1984; Cosman et al., Page and Dallman, 1991Miller et al., 19851984

M28052 M55050IL-2R b M26062Hatakeyama et al., 1989 Kono et al., 1990 Page and Dallman, 1991D11086IL-2R g D13565

Kumaki et al., 1993Takeshita et al., 1992L20048Cao et al., 1993

M74782 X64534IL-3R a

Hara and Miyajima, 1992Kitamura et al., 1991A1C2A:IL-3R b S79263

Appel et al., 1995M29855Itoh et al., 1990A1C2B:M34397Gorman et al., 1990

X69903M27959IL-4R a X52425Idzerda et al., 1990 Mosley et al., 1989 Richter et al., 1995M75914 D90205IL-5R a

Takaki et al., 1990Tavernier et al., 1991X51975 J05668IL-6R M20566/X12830

Baumann et al., 1990Sugita et al., 1990Yamasaki et al., 1988M83336 M92340gp130 M57230Saito et al., 1992 Wang et al., 1992Hibi et al., 1990

M29696 M29697IL-7RGoodwin et al., 1990 Goodwin et al., 1990

IL-8R type A M68932Holmes et al., 1991M73969IL-8R type BMurphy and Tiffany, 1991

M84746IL-9R M84747Renauld et al., 1992 Renauld et al., 1992U00672 L12120IL-10R

Ho et al., 1993Liu et al., 1994U14412IL-11R Z38102

Cherel et al., 1995 Hilton et al., 1994U23922U03187IL-12Rb1

Chua et al., 1994 Chua et al., 1995U64199IL-12Rb2 U64198

Presky et al., 1996 Presky et al., 1996X95302IL-13Ra U65747

Donaldson et al., 1998Caput et al., 1996S80963IL-13Ra % U62858

Aman et al., 1996 Hilton et al., 1996Y09328Miloux et al., 1997Y10659Gauchat et al., 1997


Table 2 (continued)

MouseHumanCytokine/growth factor Ratreceptor

U31628 U22339IL-15RGiri et al., 1995Anderson et al., 1995bU31993Il-17RYao et al., 1995b

Torigoe et al., 1997IL-18RM60468 M63122/M75862TNF-R type I M33480/M58286

Loetscher et al., 1990 Lewis et al., 1991 Himmler et al., 1990M33294 M59378Schall et al., 1990 Goodwin et al., 1991M60275/M37764Gray et al., 1990M63121/M75861Himmler et al., 1990M32315Smith et al., 1990M38549/M55994 M60469TNF-R type II U55849

Lewis et al., 1991 Bader and Nettesheim, 1996Kohno et al., 1990M59377Goodwin et al., 1991

J03143 M25764IFNgR a

Aguet et al., 1988 Kumar et al., 1989M26711Gray et al., 1989M28995Munro and Maniatis, 1989M28233Hemmi et al., 1989

U05875 S69336IFNgR b

Hemmi et al., 1994Soh et al., 1994M59820 M58288G-CSF-R

Fukunaga et al., 1990bFukunaga et al., 1990aX03663 X06368M-CSF-R X61479

Rothwell and Rohrschneider, Borycki et al., 1992Coussens et al., 19861987X68932De Parseval et al., 1993M85078GM-CSF-R a M64445Park et al., 1992Crosier et al., 1991

M38275/M59941GM-CSF-R b

Hayashida et al., 1990M77809TGFb-R type IIILopez-Casillias et al., 1991M80784Wang et al., 1991M85079TGFb-R type IILin et al., 1992


3. Techniques available for molecularimmunotoxicology

3.1. RNA isolation

The isolation of RNA is the most critical stepin the analysis of mRNA expression levels and themajority of technical failures in molecular im-munotoxicology analysis can be attributed topoor quality RNA from the original isolation.Isolated RNA molecules are highly susceptible todegradation via the activity of ribonuclease, apernicious enzyme which contaminates most labo-ratory apparatus and must be excluded from thetest RNA. We must emphasise that handling pre-cautions and ribonuclease-free apparatus and so-lutions are vital at this stage; some simple rulesfor creating such a laboratory environment havebeen laid down by Blumberg (1987). Beside qual-ity, also the reproducibility in RNA yield is ofimportance as poor reproducibility may result in alarger variability between samples within an ex-perimental group. This implies a lower sensitivityof the assay system and/or the need to increasethe number of samples per experimental group.Significant advances in RNA extraction tech-niques were made following the paper by Chirg-win et al. (1979) which described the use ofguanidinium thiocyanate to denature protein andinactivate ribonucleases. A method that we havefound suitable in our laboratory for both cellsand small pieces of tissue is that of Chomczynskiand Sacchi (1987) which uses an acid guanidiniumthiocyanate-phenol-chloroform mixture and isreadily adaptable to micro-eppendorf tubes. It isnot usually necessary to further purify the mRNAspecies from total RNA for these hybridisationstudies; in our experience this leads to irrepro-ducible recoveries. Isolation of mRNA takes ad-vantage of the polyadenylated tails of mostmRNAs by using the affinity ligand oligo (dT).Besides, this may result in additional variability.More recently, RNA isolation kits employingcolumns have been marketed and we have foundthem suitable in our laboratory. Column-basedmethods have the advantage that they do notrequire a phase separation step, in which pipettingoff the upper phase (that contains the RNA) may

introduce additional variability. Furthermore,they do not require hazardous compounds such asphenol and chloroform. Methods to directly iso-late mRNA are sometimes used in conjunctionwith RT-PCR. Isolated RNA are best stored inthe short-term as an alcoholic precipitate at −20°C, we have successfully stored them long-termat −80°C for over 6 years.

3.2. Northern blotting

Northern blotting as originally described byAlwine et al. (1977) is a technique that may causenumerous problems for inexperienced investiga-tors. RNA species are size-fractionated in a dena-turing agarose gel (usually containing formamide)and then transferred by capillary action onto anitrocellulose or nylon filter membrane which canthen be probed using labelled cDNA molecules todetect the complementary mRNA species of inter-est. The quality of RNA is clearly important(Section 3.1) and full length mRNAs are vital foridentification of the fractionated species. Initialexperiments with new cells or with a new probeshould focus on Northern blot analysis to confirmthe molecular identity of the mRNA species. Thedisadvantages of the technique are that it is notvery sensitive and that only a limited number ofsamples can be analysed on the same blot. Thetechnique also requires some experience in orderto deliver reliable reproducible results. No specificmethodology is given here but details of the basictechnique can be found in any of the standard‘cloning’ manuals. e.g. Sambrook et al. (1989).There are a variety of membranes available forNorthern transfer ranging from nitrocellulose tothe latest generation of charged nylon matrix. Inour experience the basic Northern techniqueshould be adapted to the recommendations of themembrane manufacturers in terms of hybridisa-tion solution temperatures, etc. in order toachieve the optimal signal-noise ratios. For bothNorthern blotting and dot blotting (discussed be-low) a linear relationship exists between theamount of the specific mRNA that is assayed andthe amount of signal produced (within a certainrange).


3.3. Dot or slot blotting

Whilst Northern blot techniques give confirma-tion of identity via molecular size as well as probehybridisation, it is extremely time-consuming toanalyse more than a few samples by this tech-nique. The technique of dot or slot blotting ismuch more adaptable to large sample numbers,particularly when applied to a 96 well format.Essentially the size fractionation step of theNorthern blot method is omitted and the RNAsamples are applied directly to the membranefilter via a manifold with either circular (dots) orrectangular (slots) sample holders. It is necessaryto have first confirmed molecular identity byNorthern analysis since this technique is moresusceptible to false positives due to non-specificbinding. Again it is recommended that protocolsare established which reflect the solution composi-tions and hybridisation temperatures suggested bythe manufacturers of the membranes. For bothNorthern and dot blot analysis it is necessary togenerate radiolabelled cDNA or in some cases,RNA, probes in order to detect mRNA speciesbound to the transfer membranes. There are manyways of doing this including a number of kits: inour hands the random priming technique origi-nally described by Feinberg and Vogelstein (1983)is reliable and reproducible leading to specificactivities in excess of 109 cpm/mg. Hybridisationand post-hybridisation washes should be largelyas recommended by manufacturers; however thereis some scope for experimentation in the post-hy-bridisation washing where variation in the strin-gency and temperature of the solutions can eitherguard against non-specific binding or can be usedto compensate for less than perfect hybrids, e.g.where a probe is required to detect across species.The classic visualisation and quantitation of re-sults is via autoradiography at −70°C and den-sitometry and we have obtained reproducibleresults over several years with a Shimadzu flyingspot densitometer. There is now a trend to moredirect analysis of bound radioactivity via phos-phorimagers or direct b-counting but the cost ofequipment is high.

3.4. Re6erse transcriptase-polymerase chainreaction (RT-PCR)

RT-PCR is highly sensitive and permits us tolook at modulation of cytokine expression in verylow numbers of cells or to look at the modulationof certain immunoregulatory cytokines which areexpressed at low levels within cells. A furtheradvantage is its relative ease of operation. Bytaking advantage of sequence data on cytokinesand their specific receptors (Tables 1 and 2) wecan design oligonucleotide primers for many cy-tokines for which cDNAs are not readily availableand can also design primers which will allow PCRanalysis across species barriers where there is lessthan perfect homology. The downside of RT-PCRanalysis is that it can be very difficult to quantifyexpression levels since there are many manipula-tions and numerous cycles of amplification whichcan distort small errors. This area has been exten-sively reviewed by O’Garra and Vieira (1992).Since molecular immunotoxicology focuses, inmany cases, on the relative levels of cytokineswithin cells to detect immunomodulatory events,it becomes extremely important to try to imposesome sort of quantitation on PCR results; thenext section outlines certain strategies for achiev-ing this objective.

3.4.1. Quantitation of RT-PCR productsThere are essentially three problems to consider

when attempting to impose quantitation upon anRT-PCR analysis, (a) to control for different re-covery of RNA between different samples; (b) tocontrol for differential amplification efficiency be-tween different samples; and (c) to have someassumption of linearity in the method of detectingPCR products.

Control of RNA recovery is best effected byincluding the analysis of a housekeeping genewhose steady state mRNA levels can be shownnot to vary under the conditions of the experi-ments; thus comparisons between samples can bemade by relating gene expression levels relative tohousekeeping gene expression. It has to be notedthat exposure effects on housekeeping gene ex-pression do occur (Vandebriel et al., in prepara-tion). Exposure effects on the amounts of PCR


product for housekeeping genes within a 2-foldrange is generally accepted as showing no effect.

The two most commonly used housekeepersare actin and G3PDH; neither are perfect in thattheir expression can be up or down regulated incertain scenarios but they represent a reasonablereference starting point for many experimentalconditions.

Control of differential amplification within thePCR is best effected by the use of MIMICSwhich are sections of DNA which have beenengineered to yield a PCR product using thesame oligonucleotide primers as the cDNA ofinterest but incorporate additional spuriousDNA such that the final PCR product is about100 bases larger than the cDNA of interest. Thiscompetitive PCR (Siebert and Larrick, 1992)provides an inter-tube control on each PCR reac-tion such that cDNA product can be differenti-ated from the higher molecular weight MIMICproduct on simple agarose gel electrophoresis.Careful titration of the relative amounts ofcDNA and MIMIC leads to a significant degreeof confidence in the comparison of relative geneexpression between samples. In our laboratorieswe routinely run a MIMIC for each cytokinemRNA which we subject to RT-PCR, we alsorun a housekeeping gene analysis together withits own MIMIC. Thus in an analysis of PCRproducts in a gel by densitometry, relative ex-pression is given by (OD cytokine cDNA×ODhousekeeper MIMIC) divided by (OD house-keeper cDNA×OD cytokine MIMIC).

There are many ways of analysing PCR prod-ucts, by direct densitometry of ethidium bromidestained products in a gel, by densitometry ofphotographic image of stained gel or by transferof products to hybridisation membrane and de-tection with radiolabelled probes followed by au-toradiography or direct counting. None of thesemethods are perfect and it is necessary to estab-lish the efficiency and the range of linearity foreach method in use in any given laboratory.Suffice it to say that the range of linearity isoften surprisingly poor and thus extravagantclaims of huge increases in cytokine expressiondetected by RT-PCR should always be viewedcritically.

When using MIMICs in an RT-PCR the dataobtained are the amount (generally expressed asattomoles) of a given mRNA in a sample. Theratio between the amount of mRNA encodingthe product of interest (here called X) and theamount of mRNA encoding a housekeeping en-zyme (here called H) are calculated. From this,effects on the gene expression of X can becalculated.

When the RT-PCR does not include the use ofMIMICs, the following approach can be takento assess effects on gene expression (Horikoshi etal., 1992). A serial dilution of cDNA is madeand subjected to PCR. For each dilution theamount of PCR product is plotted against theamount of cDNA (both are plotted on a linearscale). Using linear regression the slope a of theline is calculated. The correlation (r2) can beused to establish the accuracy of the line. Foreach sample this regression analysis is performedfor X and for H. The ratio of the slopes (aX

divided by aH) is then calculated for each sam-ple. From this, effects on the expression of X canbe calculated.

Some remarks should be made on this matter:1. A linear relationship between the amount of

cDNA put into the PCR and the resultingPCR product is present only in the exponentialphase of PCR. Generally, this means that thislinear relationship is found with relatively lowcycle numbers and relatively low amounts ofcDNA. This can be assessed by calculating thecorrelation of the line.

2. This type of analysis can only be made if foreach sample and for each X the efficiency ofPCR is the same. Obviously, this means thatthe number of cycles should be the same foreach X and for H (e.g. IFN-g, 27 cycles; IL-4,30 cycles; G3PDH, 24 cycles).

3. This type of analysis involves the use of slopes(with an intrinsic dose-response relationship),instead of the more commonly used analysis ofsingle measurements of PCR products (result-ing from a single amount of cDNA). Impor-tantly, analysis using slopes circumvents theproblem of how to correct for differences inthe amount of PCR product for a housekeep-ing gene. As PCRs for different genes use


different sets of primers (with different am-plification efficiencies, resulting in differentdose-response curves), corrections for differ-ent amounts of PCR product cannot bemade.

3.5. Non-radioacti6e hybridisation techniques

Since the advent of nucleic acid hybridisationassays the detection method of choice has beenthe incorporation of a radioactive 32P label intothe hybridisation probe, followed by autoradiog-raphy or direct counting. Whilst 32P detectionremains a highly sensitive method it suffers fromcertain disadvantages which become more of ahandicap as analyses move toward standardisa-tion and validation. Since 32P has a relativelyshort half-life of 14.2 days, the hybridisationprobes have a very short shelf life and need tobe re-prepared frequently. This often results inpreparation of batches with different specific ac-tivities. In fact the high specific activity of theprepared 32P probe leads to rapid radiochemi-cally induced destruction and necessitates almostimmediate use. This is not an ideal scenario forstandardisation of technique and for inter-labo-ratory comparisons. In addition 32P constitutes asignificant health hazard unless strictly containedand generates major disposal problems such thata number of research organisations do not allow32P labelling methods into their laboratories.

Non-radioactive hybridisation techniques (re-viewed in Kricka, 1995) offer considerablepromise for the future. Especially hybridisationassays that are based on digoxigenin-labeled nu-cleotides and a dioxetane substrate have beenreported to be highly sensitive (Lanzillo, 1991;Engler-Blum et al., 1993). The advantage ofthese techniques is that probes can be preparedin bulk and stored for prolonged periods with-out detectable loss of activity, also there arevery few problems associated with handling theprobes which can be prepared in a routine non-specialised laboratory. However, the sensitivityof non-radioactive hybridisation is as yet insuffi-cient for routine measurement of cytokinemRNA levels.

4. Applications and specific examples of molecularimmunotoxicology

In the following sections, specific examples ofmolecular biological analysis of immunomodula-tion are given, grouped under in vitro, ex vivo orin vivo effects.

4.1. In 6itro analysis of immunomodulation

4.1.1. BiostimBiostim (RU 41740) is an extract from Kleb-

siella pneumoniae which is substantially composedof two high molecular weight glycoproteins of 350and 95 kDa. This preparation has been shown tohave immunomodulatory activity both in animals(Takada et al., 1982) and in humans (Capsoni etal., 1985). In a series of experiments (Meredith etal., 1990) we were able to demonstrate that inquiescent macrophage cell populations, steadystate levels of mRNAs for IL-1a and IL-1b weredramatically increased in response to 1 mg/mlBiostim. Further experiments showed that levelsof Biostim as low as 1–10 pg/ml could elevatesteady state levels of mRNAs for IL-1a, IL-1b,IL-6 and TNF-a whereas there was no effect onthe expression of the housekeeping gene, actin.This preparation was shown to be approximately100-fold more potent than a standard preparationof lipopolysaccharide. In this system the expres-sion of all cytokine mRNAs was transient, peak-ing after 1–3 h and returning to basal levels after24 h. These findings correlate well with docu-mented effects on IL-1 and TNF-like secretion inhuman monocyte populations (Sozzani et al.,1988) and suggest that this type of in vitro analy-sis may be used both as a rapid screen for im-munomodulatory activity and to suggestmechanisms of action.

4.1.2. Cyclosporin AThe immunosuppressive drug Cyclosporin A

(CsA) exerts its action by binding to cyclophilin.Binding of the CsA/cyclophilin complex to theprotein phosphatase calcineurin results in inacti-vation of calcineurin. As a consequence, dephos-phorylation of the cytosolic subunit of the nuclearfactor of activated T-cells (NF-AT), necessary for


translocation of this transcription factor to thenucleus, is blocked. Absence of this translocationresults in a failure to activate the genes regulatedby NF-AT, such as IL-2 (Cai et al., 1996; Ho etal., 1996). CsA treatment of Con A stimulatedmouse spleen cells reduced mRNA expression ofIL-2, IL-3, IFN-g, GM-CSF, and TNF-a, but notIL-5, IL-6, and IL-10, whereas mRNA expressionof TGF-b and IL-1b was enhanced (Han et al.,1995). In a series of experiments in our laboratorywe were able to show that CsA can inhibit theexpression of IL-2 mRNA within a murine mixedlymphocyte culture (Meredith and Scott, 1994).The effects were dose-dependent from 1–100 ng/ml with a maximal 90% inhibition of IL-2 steadystate levels at the highest dose. Similar effectswere seen in a rat mixed lymphocyte culture. Thuswithin an in vitro model of T cell activation theeffects of an immunosuppressive drug can beclearly demonstrated, illustrating the potential ofmolecular immunotoxicological analysis to notonly screen for activity but also, in certain cases,to give indications of mechanism of action.

4.1.3. Tributyltin oxideThe organotin compound tributyltin oxide

(TBTO) was used extensively as a molluscicideand in anti-fouling paint: its ability to accumulatein molluscs has led to fears of its entry into thefood chain. TBTO is well known to exert a selec-tive immunotoxicity in a rat model (Vos et al.,1984, 1990) although the precise molecular mech-anism of action remains unknown. Within ourlaboratory we were able to show that in vitroexposure to TBTO within a rat mixed lymphocytereaction (model of T cell activation) resulted in adose-dependent inhibition of expression of IL-2receptor mRNA levels within cells from the MLR(Meredith et al., 1991a). These effects were dose-dependent between 0.1 and 10 nM; higher concen-trations proving cytotoxic. We also showedinhibition of expression of serine protease mRNAat the same concentrations within the MLRs.Conversely, cultured macrophages were less sensi-tive to immunomodulation of cytokine expressionby TBTO, although minor effects were seen onIL-1b expression in Biostim activatedmacrophage cultures. Thus in this case, although

the mechanism of TBTO immunotoxicity is not adirect effect on cytokine expression, careful moni-toring of the expression of cytokines within invitro models of immune activation (cytokineprofiling) can lead to conclusions about the natureof the immunotoxicity.

4.2. Ex 6i6o analysis of immunomodulation

4.2.1. BiostimAs described previously in Section 4.1.1, Bios-

tim is an immunomodulatory drug preparationwhich has been shown to have immunomodula-tory activity to the murine immune system (Woodand Moller, 1984, 1985) and to humanlymphocytes both in vivo and in vitro (Meroni etal., 1987). In our laboratory we studied the effectsof Biostim on the expression of cytokine mRNAsin murine peritoneal macrophages following in-traperitoneal administration at a dose of 100 mg/kg (Meredith et al., 1991b). In vivo, residentperitoneal macrophages in C3H mice express lowlevels of cytokine mRNAs; however followingexposure to Biostim, expression of mRNAs forIL-1a, IL-6 and TNF-a was transiently stimu-lated, with up to 100-fold elevation in the steadystate levels of mRNA transcripts at 6 h post-dos-ing. A similar elevation was seen in the level oftranscripts for IL-1b, but the expression was morepersistent, detectable up to 24 h post-dosing. Thisresult contributes further evidence to the argu-ment that analysis of altered cytokine mRNAexpression in defined immune cell populationstreated in vitro correlates with the effects of im-munomodulatory agents in vivo (Meredith andScott, 1994).

4.2.2. Tributyltin oxideAs outlined in Section 4.1.3, tributyltin oxide

(TBTO) is known to induce immunotoxicity in arat model, causing thymic atrophy, lymphocytedepletion in spleen and lymph nodes and in-creased serum IgM and decreased serum IgG(Krajnc et al., 1984) and suppression of thymus-dependent immune responses (Vos et al., 1984).The major target organ is known to be the thy-mus where there appears to be a direct action oncortical thymocytes (De Waal et al., 1993). In a


series of experiments in our laboratories, malerats were exposed to TBTO at doses of 5, 20 or 80mg/kg diet for 6 weeks, the top dose being chosenfor overt immunotoxicity. Molecular cytokineanalysis of Con A stimulated spleen cells from theexposed animals showed that there was a dose-de-pendent decrease in IL-2 receptor mRNA levelsfrom 5 mg/kg diet, a dose-dependent increase inIFN-g mRNA levels from 20 mg/kg diet and anincrease in IL-2 mRNA at the top dose (Meredithet al., 1994; Vandebriel et al., 1998a). This sug-gests that one of the early events in TBTO im-munotoxicity may be a down-regulation of IL-2receptor expression and this may be involved inthe sequence of events, largely uncharacterised,which leads to an impediment in thymocyte matu-ration and subsequent thymic atrophy.

4.3. In 6i6o analysis

4.3.1. OzoneAirway inflammation seems to be a common

mechanism in a number of health effects resultingfrom acute ozone exposure, like airway tissueinjury, hyperresponsiveness, and exacerbation ofrespiratory diseases. Some of these effects may beindicative for the onset of long-term effects whichmay result from repeated exposure. A limitednumber of studies on acute ozone inhalation hasshown increased synthesis and release of inflam-matory cytokines in airways. Effects of repeatedexposures to ozone are, however, unknown. Theaim of this study was to compare the pulmonarygene expression of a large panel of (pro-)in-flammatory cytokines following acute and re-peated exposure to ozone.

Semiquantitative RT-PCR showed that acuteinhalation of ozone in rats induced increasedmRNA levels of IL-1b, IL-6, KC, and MIP-2,whereas TNF-a, TGF-b, MCP-1, and fibronectinmRNA levels remained unchanged. Compared tothis acute inflammation, repeated ozone exposureinduced a different pattern of gene expression.The mRNA level of IL-6 was increased and offibronectin was decreased, whereas IL-1b, KC,MIP-2, TNF-a, TGF-b, and MCP-1 mRNA lev-els remained unchanged. Repeated ozone expo-sure has previously been shown to result in airway

remodelling, increase of inflammatory cells, andcollagen deposition, indicative for (an onset of)fibrotic lesions. We conclude that: (1) acute andrepeated exposure to ozone result in differences inpulmonary gene expression of inflammatory cy-tokines; and (2) ozone-induced airway tissue(fibrotic) lesions are not accompanied by in-creased expression of possibly involved cytokines(Van Bree et al., 1996).

4.3.2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD)

The immune system has been identified as oneof the most sensitive targets for the toxicity of theenvironmental contaminant 2,3,7,8,-tetra-chlorodibenzo-p-dioxin (TCDD). The thymus isespecially sensitive to the toxic action of TCDD.Much of its toxicity is initiated by binding to thearyl hydrocarbon receptor (AhR), an intracellularreceptor that functions as a ligand-activated tran-scription factor. Exposing mice to TCDD resultsin decreased host resistance to infectious diseasesand suppressed cell-mediated and thymus-depen-dent humoral immune responses (Vos and Luster,1989; Kerkvliet and Burleson, 1994).

TCDD exposure of C57Bl/6 mice results in adose-dependent suppression of the in vivo cyto-toxic T-cell (CTL) response to P815 mastocytomacells (De Krey and Kerkvliet, 1995). This suppres-sion is mediated through the AhR (Kerkvliet etal., 1990). In this model, until day 5 followingP815 injection, cytokine production by spleencells was induced normally. From day 5 andonwards the levels for IFN-g, IL-2, and TNF-afailed to increase in exposed animals. IL-4, IL-6,and IL-1b levels, however, remained unaffectedby TCDD exposure. This differential effect ofTCDD on cytokine production corresponded toexposure effects on alloantibody isotype, namelythe abrogation of IgG2a levels (induced by IFN-g) and a much smaller effect on IgG1 levels(induced by IL-4). IFN-g and IL-2 were found tobe produced only by the CD8+ subset of spleencells, at least between day 4 and 7 post-injection.In sum, this suggests that cytokines produced byTC1 cells are suppressed upon TCDD exposure invivo (Kerkvliet et al., 1996).


This example shows that in this model alteredcytokine levels: (1) are able to detect immunotoxi-city due to TCDD exposure; and (2) provideinsight into possible mechanisms of TCDDimmunotoxicity.

4.3.3. AzathioprineThe immunosuppressive drug azathioprine

(AZP) is a 6-mercaptopurine analogue that pre-sumably exerts its effects on T-cell mediated re-sponses by interference with de novo synthesis ofpurines (Caspritz and Hadden, 1987). In bothmacrophage and lymphocyte cultures, AZP inhib-ited the expression of all mRNAs studied, includ-ing the housekeeping gene actin at aconcentration of 1 mg/ml (Meredith et al., 1991b).This non-specificity of action was confirmed usingin vivo analysis (Meredith and Scott, 1994) wherefollowing repetitive administration of AZP toPVG rats over 28 days (2 mg/kg per day), theexpression of both IL-2 and actin mRNAs wasalmost totally inhibited within the isolated thymo-cytes. Thus we can conclude that the immunotox-icity of AZP is non-specific and results from acytotoxic mode of action.

4.3.4. Dinitrochlorobenzene (DNCB) andtrimellitic anhydride (TMA)

Two CD4+ T helper (TH) cell subsets, desig-nated TH1 and TH2, exist. Their identificationhas greatly improved the understanding of theregulation of immune effector functions, not inthe least on Type I and Type IV hypersensitivityresponses. These TH subsets cannot be differenti-ated on the basis of a cell surface marker. Theyproduce, however, defined patterns of cytokinesthat lead to strikingly different T-cell functions.Roughly speaking, TH2 cells are more efficientB-cell helpers, especially in the production of IgE,whereas TH1 mediate delayed-type hypersensitiv-ity (DTH). In addition, they cross-regulate bymutually antagonistic cytokines.

Most resting T-cells mainly produce IL-2 onfirst contact with antigen, and differentiate withina few days into cells producing multiple cytokines,such as IFN-g and IL-4. The balance of cytokinesthat are present in the microenvironment duringearly stages of the immune response, in particular

IFN-g and IL-4 may greatly influence the direc-tion of the immune response. IFN-g augmentsdevelopment of TH1-type responses and IL-4 pro-motes differentiation of TH2 cells. Macrophagesand NK cells are a major source of IFN-g,whereas mast cells and basophils are a majorsource of IL-4. gd T-cells recognise antigen (Ag)differently from ab T-cells. They do not requireAg processing for recognition and are able torecognise nonpeptide ligands (Chien et al., 1996).These cells are capable of producing both IL-4and IFN-g.

Chemicals differ with respect to the type ortypes of allergic reactions they will elicit. Dear-man et al. have shown that chemicals known tocause respiratory hypersensitivity induce in micepreferential TH2-type responses and IgE anti-body. In contrast, chemical contact allergenswhich are known or suspected not to cause res-piratory hypersensitivity, induce instead immuneresponses consistent with a selective activation ofTH1 cells (Dearman and Kimber, 1991, 1992;Dearman et al., 1992a,b,c; Kimber and Dearman,1992). Compounds that preferentially induceTH1- or TH2-type responses can be identified bytheir cytokine production profiles, namely IFN-g(produced by TH1) with a concomitant lack ofproduction of IL-4 and IL-10 (produced by TH2)by chemical contact sensitizers and the reverse forchemicals known to cause respiratory hypersensi-tivity. In order to measure IL-4, lymph node cellshave to be stimulated with Con A (Dearman etal., 1994), whereas IFN-g and IL-10 do not re-quire this stimulation (Dearman et al., 1995,1996).

The compound dinitrochlorobenzene (DNCB)that induces TH1-type responses induces a signifi-cantly higher level of IFN-g mRNA than thecompound trimellitic anhydride (TMA) that in-duces TH2-type responses. Both DNCB andTMA induce IL-4 expression (Vandebriel et al.,1998b). This suggests that for IFN-g increasedmRNA levels are paralleled at the protein level,whereas for IL-4 this is not the case. In conclu-sion, cytokine measurements using molecular bio-logical methods can be very useful to assess thedirection of immune responses (TH1 or TH2)upon sensitisation. Thus, compounds that show


allergenicity (e.g. by inducing lymphocyte prolifer-ation) can be classified as skin allergen or respira-tory allergen.

4.3.5. Mercury chloride (HgCl2)A well-known model for chemical-induced au-

toimmunity is HgCl2-induced autoimmunity inBrown Norway (BN) rats. This syndrome results inproduction of a number of autoantibodies and anenhanced level of serum IgE. Furthermore, necro-tising vasculitis, predominantly of the caecum, isobserved. The syndrome is T-cell dependent, andthe enhanced level of serum IgE suggests thepredominant involvement of Th2 cells. HgCl2treatment of Lewis rats does not result in any ofthe described immune alterations or pathologies.

The observation of caecal vasculitis as early as24 h after HgCl2 administration suggested a role inthe disease process of a cell type different from theones involved in the T-cell response. Oliveira et al.(1995) showed that HgCl2 treatment of mast cellsfrom BN rats enhanced the release of serotoninupon IgE crosslinking in vitro. This was observedto a much lesser extent for mast cells from Lewisrats. Similar observations were made with twoother chemicals that induce a similar type ofautoimmunity, i.e. gold salts and D-penicillamine.

In the same study the authors showed that HgCl2treatment of BN mast cells resulted in IL-4 mRNAexpression. This was not observed for mast cellsfrom Lewis rats. It had previously been suggestedthat IL-4 from mast cells provide the initial sourceof IL-4, in order for Th2 responses to develop(Romagnani, 1992). In vivo, HgCl2 treatment ofBN rats results in IL-4 mRNA production in thespleen within 1–2 days (Gillespie et al., 1995).

In conclusion, compounds that induce autoim-munity in BN rats in vivo can exert a direct effecton mast cells in vitro to enhance mediator releaseand induce cytokine production. Furthermore, it ishypothesised that mast cell degranulation is re-sponsible for the early vasculitis observed. Finally,a role for IL-4 produced by mast cells in theinitiation or augmentation of the cellular immuneresponse resulting in chemical-induced autoimmu-nity is hypothesised.

5. Summary

Molecular immunotoxicology involves the anal-ysis of altered expression of cytokines and relatedmolecules within defined immune cell populationsas an indicator of immunomodulation. There area variety of techniques for identifying and quan-tifying cytokine mRNAs including hybridisationtechnology and RT-PCR. Analyses can be per-formed on isolated cell populations following invitro exposure to immunomodulatory agent or oncells or tissues taken ex vivo. Molecular im-munotoxicological analysis has led to a betterunderstanding of mode of action of certain im-munotoxicants, e.g. TBTO, and has definedparameters for cytokine profiling which will lead tothe development of screens for immunomodulatorypotential (Meredith and Miller, 1994) and lead toa more integrated approach within in vitro im-munotoxicology (Meredith and Miller, 1997). Per-haps the most significant advantage of thetechnique is the potential to not only screen forimmunomodulatory activity but also to give indi-cations of the mechanism of action. This appliesnot only to the drugs and chemicals cited in thisreview but also to other areas of immune pathol-ogy, e.g. in man-made mineral fibre induced lungdisease (Meredith, 1995).

For the future, it is likely that techniques will bemade more robust and that quantitation will be-come more accurate and precise. Greater under-standing of the role of cytokines within immunemodels of activation will lead to identification ofkey markers of immune dysfunction which can beincorporated into routine cytokine profile screen-ing. Finally, combinations of techniques are likelyto become more important in diagnosis of im-munotoxicity, e.g. coupling of in situ hybridisationfor intracellular mRNA with cell surface markeranalysis and correlation of aberrant cytokine ex-pression with changes in immune functionality.

Acknowledgements

Work in the authors’ laboratories has beensupported by the EU STEP program cCT91-


0142 and by the Health and Safety Executive,UK. We thank Dr Joseph G. Vos for criticalreading of the manuscript and Dr D. Donaldsonfor discussion.

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Altered cytokine (receptor) mRNA expression as a tool in immunotoxicology

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