use of transgenic animals to investigate drug hypersensitivity

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Toxicology 158 (2001) 75–83 Use of transgenic animals to investigate drug hypersensitivity Rene Moser a , Valerie Quesniaux a , Bernhard Ryffel b, * a Institute of Biopharmaceutical Research, Matzingen, Pharma No6artis AG, Basel, Switzerland b Department of Immunology, Uni6ersity of Cape Town, Cape Town, South Africa Abstract Hypersensitivity reactions to drugs and environmental agents are often due to exaggerated humoral (Th 2 ) or cell mediated (Th 1 ) immune responses with typical cytokine profiles. Overexpression of Th 2 cytokines, such as IL-4, IL-5 or IL-13 in mice, enhances an IgE antibody mediated response, while deletion of these cytokines attenuates and/or prevents allergic responses. Conversely, modulation of Th 1 cytokine gene expression may affect cell-mediated immune responses. Therefore, cytokine transgenic mice are used as investigative tools to study potential chemicals and/or drug allergies. In addition to cytokines and chemokines, other factors are important for the development of allergic responses, such as IgE, Fc receptors, vasopressin and several other factors, which can be tested in transgenic mice. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allergy; Asthma; Eczema; Cytokine transgenic mice www.elsevier.com/locate/toxicol 1. Introduction Hypersensitivity reactions to environmental agents, food, chemicals and drugs are common in man, and their incidence seems to be increasing (Woolcock and Peat, 1997; Howarth, 1998). Basi- cally, two pathophysiological mechanisms for the development of hypersensitivity reactions can be distinguished: (1) antibody-dependent degranula- tion of mast cells in sensitized individuals (IgE); and (2) T cell-dependent, inflammatory responses (Burns and Gaspari, 1996; Kimber et al., 1998a,b; Coleman and Blanca, 1998). The antigen may be a protein, a drug/chemical, or a metal. Typical clinical features of an IgE-dependent allergic reac- tion comprise urticaria, erythema, asthma and in severe cases, even shock (Bernstein and Bernstein, 1994). Dermal exposure to allergens may result in chronic cutaneous hypersensitivity reactions known as eczema. The question of why an inert protein causes allergy in some, but not all individuals indicates that host factors play an important role. In man, several genes have been identified on chromosome 5 (IL-3, IL-4, IL-5, IL-9, IL-13), 6 (HLA-D and TNF) and 12 (IFN-g, stem cell factor, STAT6), which are associated with allergy (Barnes and * Corresponding author. Present address: CNRS Orleans, CDTA, UPS 44, 3B rue de la Ferollerie, 45071 Orleans, Cedex 02, France. Tel.: +33-238-255452; fax: +33-238-255435. E-mail address: [email protected] (B. Ryffel). 0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0300-483X(00)00411-X

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Toxicology 158 (2001) 75–83

Use of transgenic animals to investigate drughypersensitivity

Rene Moser a, Valerie Quesniaux a, Bernhard Ryffel b,*a Institute of Biopharmaceutical Research, Matzingen, Pharma No6artis AG, Basel, Switzerland

b Department of Immunology, Uni6ersity of Cape Town, Cape Town, South Africa

Abstract

Hypersensitivity reactions to drugs and environmental agents are often due to exaggerated humoral (Th2) or cellmediated (Th1) immune responses with typical cytokine profiles. Overexpression of Th2 cytokines, such as IL-4, IL-5or IL-13 in mice, enhances an IgE antibody mediated response, while deletion of these cytokines attenuates and/orprevents allergic responses. Conversely, modulation of Th1 cytokine gene expression may affect cell-mediated immuneresponses. Therefore, cytokine transgenic mice are used as investigative tools to study potential chemicals and/or drugallergies. In addition to cytokines and chemokines, other factors are important for the development of allergicresponses, such as IgE, Fc receptors, vasopressin and several other factors, which can be tested in transgenic mice.© 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Allergy; Asthma; Eczema; Cytokine transgenic mice

www.elsevier.com/locate/toxicol

1. Introduction

Hypersensitivity reactions to environmentalagents, food, chemicals and drugs are common inman, and their incidence seems to be increasing(Woolcock and Peat, 1997; Howarth, 1998). Basi-cally, two pathophysiological mechanisms for thedevelopment of hypersensitivity reactions can bedistinguished: (1) antibody-dependent degranula-tion of mast cells in sensitized individuals (IgE);and (2) T cell-dependent, inflammatory responses

(Burns and Gaspari, 1996; Kimber et al., 1998a,b;Coleman and Blanca, 1998). The antigen may bea protein, a drug/chemical, or a metal. Typicalclinical features of an IgE-dependent allergic reac-tion comprise urticaria, erythema, asthma and insevere cases, even shock (Bernstein and Bernstein,1994). Dermal exposure to allergens may result inchronic cutaneous hypersensitivity reactionsknown as eczema.

The question of why an inert protein causesallergy in some, but not all individuals indicatesthat host factors play an important role. In man,several genes have been identified on chromosome5 (IL-3, IL-4, IL-5, IL-9, IL-13), 6 (HLA-D andTNF) and 12 (IFN-g, stem cell factor, STAT6),which are associated with allergy (Barnes and

* Corresponding author. Present address: CNRS Orleans,CDTA, UPS 44, 3B rue de la Ferollerie, 45071 Orleans, Cedex02, France. Tel.: +33-238-255452; fax: +33-238-255435.

E-mail address: [email protected] (B. Ryffel).

0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0300-483X(00)00411-X

R. Moser et al. / Toxicology 158 (2001) 75–8376

Marsh, 1998; Holloway et al., 1999). Genetic link-age analyses demonstrated that the developmentof hypersensitivity is under the control of multiplegenes, the individual role of which is unknown.Experimental animal models, however, demon-strated significant roles of most of the gene prod-ucts identified above, alone or in combination. Inview of the complex genetic control of allergicresponses and our limited understanding, hyper-sensitivity reactions are impossible to predicttoday.

Small chemicals and drugs may have a reactivegroup allowing covalent binding to a protein andhence can be recognized by the immune system asforeign (Coleman and Blanca, 1998). In the ab-sence of reactive groups, metabolic activation maygenerate haptens that sensitize T cells (Griem etal., 1999; Ewens et al., 1999). Recent evidencesuggests that covalent binding may not be a pre-requisite (Zanni et al., 1997; Schnyder et al.,1998).

Concomitant infections, such as bacterial infec-tions, may reduce the incidence of allergic re-sponses (Erb et al., 1998), while parasiticinfections increase it (Erb, 1999; Adams et al.,1999). In the case of mycobacterial infections, orfollowing immunization with BCG, which is astrong Th1 response inducer, an inverse associa-tion between tuberculin responses and atopic dis-orders has been reported (Cookson and Moffatt,1997; Shirakawa et al., 1997). Therefore, bacterialinfections may bias the immune system to a pre-dominantly Th1 response and reduce allergicreactions.

2. Role of cytokines and chemokines

The immune system has essentially two ways ofreacting to foreign substances/drugs: either thehost produces antibodies of the IgE type (hu-moral response mediated by Th2 lymphocytes) orshows a cell mediated immune (Th1) response, asdescribed in mouse and man (Abbas et al., 1996;Romagnani, 1997).

IL-12 and IL-18 produced by antigen-present-ing cells, e.g. macrophages and dendritic cells, andIFN-g, LTb and IL-2 from activated T cells,

allow the differentiation of Th1 cells. The Th1

response comprises delayed-type hypersensitivity(DTH) reaction, granuloma and eczema. IL-4,IL-5 and IL-13 from basophils and/or NK and Tcells promote Th2 cell differentiation, resulting inantibody production manifested in allergic skinreactions, asthma and allergic shock.

Allergic drug reactions fall into both categories.The Gell and Coombs’s classification includesfour types (I–IV) of immunopathological reactionmechanisms. Type I reaction is identical with theTh2 response, while type IV coincides with theTh1 reaction. Cytotoxic antibodies (type II) andimmune complex-induced reactions (type III) canbe considered as special forms of antibody-medi-ated immune reactions resembling a Th2 response,and they are also involved in hypersensitivityreactions to drugs. Conventional drugs (chemi-cals) can act as haptens and act only upon bind-ing to a protein, is an immunogenic antigenformed that is presented in the context of MHCby professional antigen presenting cells, such asdendritic cells or macrophages (Grabbe andSchwarz, 1998; Kimber et al., 1998a,b). TNF andIL-1 are apparently required for the differentia-tion and migration of dendritic cells to the re-gional lymph nodes (Kimber et al., 1998a,b),while IL-10 has an inhibitory role (Wang et al.,1999). The requirement of dendritic cells in aller-gic reactions has been conclusively demonstratedby eliminating dendritic cells using myeloid spe-cific thymidine-kinase transgenic mice and gancli-covir (Lambrecht et al., 1998). The factorsdeciding whether a prevailing Th1 or Th2 immuneresponse is induced, are debatable. This issue iscomplex and poorly understood. The initial cy-tokine(s) produced in DC and other cells (mastcells and NK cells) appear to determine the differ-entiation of so-called Th0 cells into either Th1 orTh2 cells.

Bacterial and parasitic antigens are contrastingexamples inducing either Th1 or Th2 responses.Mycobacterial cell walls are often used as adju-vant and are potent Th1 inducers, while schistoso-mal egg antigens (SEA) are potent Th2 inducers.Besides the nature of the antigen, the dose androute of presentation can also affect the Th1/Th2

differentiation. Recently another family of peptide

R. Moser et al. / Toxicology 158 (2001) 75–83 77

factors with chemotactic functions, known as thechemokines, has been shown to have a regulatoryrole in the process of Thl/Th2 differentiation andhence in Th1 vs Th2 responses (Baggiolini et al.,1997).

This review focuses on investigating the role ofcytokines in hypersensitivity reactions. As im-munological and genetic tools are available, andhypersensitivity reactions develop in mice (Kim-ber and Dearman, 1997; Vargaftig, 1999), thediscussion will focus on genetic models in mice.

The selection of inbred strains already has asubstantial impact on the Th1/Th2 response pat-tern: for example, C57/BL6 mice have a preferen-tial Thl response, while Balb/c mice tend todevelop a Th2 response (Sun et al., 1997). Fur-thermore, nature has created its own discrete mu-tations in several genes of interest, e.g. stem cellfactor, CSF-l, TLR4, FAS, lpr, and many others.Finally, overexpression and deletion of genes isnow possible in mice by recombinant technology,which allows us to test for the role of a given genein a pathophysiological process.

3. Transgenic mice

The technique of modifying the mouse genomewas developed in the last 10 years. Injection ofDNA/gene into oocytes followed by implantationof the transfected oocyte into the uterus of pseu-dopregnant mice results in a progeny of miceoverexpressing the randomly inserted gene. Incontrast, homologous recombination in embry-onic stem cells is used to generate mice deficient(knock-out, KO) for a specific gene. The latterinvolves generation and selection of mutant em-bryonic stem cells, their injection into a blasto-cyst, implantation into the uterus and breedingthe progeny to homozygosity (Burki and Leder-mann, 1995). A vast number of mutant mice hasbeen generated (reviewed in Mak, 1998).

Both types of mutants — mice overexpressingor with deletions of specific genes (KO) — provedto be valuable tools for the study of pharmacol-ogy, toxicology and disease (Wei, 1997; Ryffel,1997; Lovik, 1997; Eynon and Flavell, 1999). The

Table 1Cytokine deficient mice — Th1 versus Th2

ReferencePhenotypeKO Th1/Th2

Th2Reduced host resistanceIFN-g Dalton et al., 1993Huang et al., 1993

Reduced DTH Cooper et al., 1993Th2 Sadlack et al., 1993Colitis, anemiaIL-2

Reduced host resistanceIL-12 Th2 Magram et al., 1996Cooper et al., 1997

IL-18 Th2Reduced host resistance Wei et al., 1999IL-12/18 Th2Reduced host resistance Takeda et al., 1998

Paspararakis et al. 1996Th2TNF Reduced host resistanceMarino et al., 1997

No lymph nodesLT Th2 De Togni et al., 1994Sadl ack et al., 1993IL-10 ColitisBerg et al., 1995; Wang et al., 1999

IL-3 Th1Reduced DTH Mach et al., 1998Reduced IgE and DTHIL-4 Th1 Kopf et al., 1993

IL-5 Reduced eosinophils Th1 Kopf et al., 1993Reduced IgE, IL-4, IL-5 McKenzie et al., 1998IL-13 Th1

Cohn et al., 1999Th1IL-4Ra Reduced BHRBrombacher et al., 1998

IL-4/13 Reduced parasite response Th1 McKenzie et al., 1999

R. Moser et al. / Toxicology 158 (2001) 75–8378

main phenotypic features of Th1 and Th2 cytokineKO mice are briefly reviewed in Table 1.

3.1. IFN-g KO

Impaired activation of phagocytes and defectiveproduction of microbiocidal nitric oxide results inreduced host resistance to microbial antigens (Dal-ton et al., 1993; Huang et al., 1993). The cell-medi-ated immune response to microbial antigen andchemical irritants is reduced (Saulnier et al., 1995).

3.2. IL-2 KO

Surprisingly IL-2 KO mice initially develop nor-mally and have a functional immune system (Sad-lack et al., 1993). However, within the first monthsof life, a severe autoimmune disorder develops(ulcerative colitis and anemia).

3.3. TNF, LTa KO

These mice confirmed that TNF plays an essen-tial role in microbial resistance and DTH, whileLTa is required for the normal development of thelymphoid organs (De Togni et al., 1994; Eugster etal., 1996; Pasparakis et al., 1996; Marino et al.,1997).

3.4. IL-12 KO

Mice deficient in IL-12 have impaired IFN-gproduction and reduced Th1 responses: DTH re-sponses were significantly reduced and IL-4 secre-tion increased (Magram et al., 1996). IL-18 may inpart compensate for the absence of IL-12.

3.5. IL-4 KO

These mice have a normal T and B cell develop-ment, but IgG1 and IgE serum levels are reduced.In addition, they mount no antigen-specific IgEresponses to the nematode Nippostrongylusbrasiliensis and a predominantly Th1 cytokine re-sponse (Kuhn et al., 1991; Kopf et al., 1996). Areduced cutaneous delayed-type hypersensitivityto the hapten trinitrochloro-benzene has beendemonstrated (Dieli et al., 1999).

3.6. IL-5 KO

These mice have reduced eosinophilia in re-sponse to nematode infections (Kopf et al., 1996).

3.7. IL-13 KO

IL-13 deficient mice have reduced IgE and IL-4responses (McKenzie et al., 1998). The IL-13 andthe IL-4/IL-13 double deficient mouse (McKenzieet al., 1999) as well the IL-4-Ra deficient mouse(Cohn et al., 1999; Brombacher et al., 1999), thathas an identical functional phenotype, as the IL-4Ra chain is required for both IL-4 and IL-13signaling, demonstrated an essential and novelrole of IL-13 in allergic asthma and parasiticdisease. The role of IL-13 in the drug hypersensi-tivity reaction has not been explored, but mayopen new insights.

4. Cytokine transgenic mice

IL-4 (Tepper et al., 1990) and IL-5 (Dent et al.,1990; Tominaga et al., 1991) overexpressing micemay be of special interest as they mimic a Th2 typedisorder that may be enhanced by allergens. Usinga lung epithelial promotor, IL-5 (Lee et al., 1997),IL-13 (Zhou et al., 1999) and IL-9 (Dong et al.,1999) transgenic mice develop pathologic changescharacteristic of asthma and may provide a sensi-tive model to evaluate the potential efficacy andsafety of new drugs. The cutaneous allergic re-sponse to dinitrofluorobenzene is significantly in-creased in IL-5 transgenic mice suggesting theiruse in predicting the Th1 inducing potential ofchemicals/drugs (Nagai et al., 1999).

5. Second generation transgenic mice

A major limitation of the first generation mu-tant mice is that the gene is either randomlyinserted into the genome in the case of transgenicmice, or is completely inactivated as in KO mice.If the targeted gene plays a role in the embryonicdevelopment, inactivation may be embryo-lethalor result in abnormal development, as shown for

R. Moser et al. / Toxicology 158 (2001) 75–83 79

example in LTa KO mice, which lack lymphnodes (De Togni et al., 1994). Therefore, strate-gies were developed to target alterations by usingthe cre/lox approach (Rajewsky et al., 1996). Thisimplies the insertion of loxP sites on both sides ofthe gene of interest by homologous recombination(flaxed gene). The lox sites are recognised by thecre recombinase, which cuts the inserted genesegment at the loxP sites. In vivo, the eliminationof the targeted gene is performed by mating theflaxed mice with cre transgenic mice. Tissue spe-cificity is achieved by using cre transgenic mice inwhich cre is expressed under a tissue-specific pro-motor. This allows, for example, the inactivationof genes in macrophages, lymphocytes, or lungepithelial cells. These tissue-specific KO mice willbe interesting tools to investigate cutaneous andpulmonary hypersensitivity reactions.

The next step of sophistication is the use of aninducible promoter, such as the tetracyclin (tet)promoter. Administration of tetracyclin in tet–crctransgenic mice allows controlled gene activationor deletion in adult mice. Furthermore, genes canbe introduced into the mouse genome by the sametechnique of homologous recombination. The so-called ‘KO-in’ mice can express human genes,which may useful in certain experimental condi-tions (Taneja and David, 1998).

6. Mouse models to test drug hypersensitivity

The following experimental approaches can beused to test the allergic potential of chemicals/drugs.

6.1. Systemic immune response after immunisation

The antigen is injected parenterally with orwithout an adjuvant. Depending on the choice ofadjuvant, a Th1 (Freund’s complete adjuvant) orTh2 response (Alum) is favoured.

6.1.1. Determination of antibody responseTypically (IgG isotypes and IgE levels) IgG2a

and IgG2b antibodies are induced in Th1 re-sponses, while IgG1 and IgE are produced in Th2

responses.

6.1.2. Restimulation of lymphocytes of locallymph nodes or splenocytes with antigen

Determination of proliferation, cytokine tran-scripts or proteins (RNA, ELISA).

6.2. Delayed-type hypersensiti6ity reaction (DTH)

In response to cutaneous challenge of sensitizedmice, local oedema and inflammation developswithin 24 h. The reaction is driven by Th1 cellsand depends on the production of IL-12, TNFand IFN-g. Langerhans cells process the antigen/chemical, differentiate to mature dendritic cellsand migrate from the skin under the influence ofIL-1 and TNF to the regional lymph node, wherethey exert their antigen presenting function, re-sulting in the recruitment and activation of effec-tor T cells (Kimber et al., 1998a,b). Recently arole for Th2 cells and cytokines has been shown incertain forms of atopic dermatitis (Grewe et al.,1998), demonstrating that the Th1/Th2 paradigmhas limitations (Kitagaki et al., 1999). In addition,a clear distinction has to be made between andirritant/toxic vs hypersensitivity reactions, such ascroton oil inflammation, which does not needsensitization.

A typical protocol is as follows: local challengeby epicutaneous application or intradermal injec-tion (footpad or ear) 10 days after initial immu-nization by injecting antigen in Freund’s adjuvant(subcutaneously). The footpad or ear swelling isassessed at 24 and 48 h after local challenge witha micrometer. This can be combined with analysisof the response in the regional lymph node(weight, cell number, proliferation, cytokinerelease).

6.3. Bronchial hyperreacti6ity (BHR)

For the investigation of respiratory hypersensi-tivity reactions, ovalbumin is commonly used,although other proteins induce identical responses(Morone, 1998; Wills-Karp, 1999). Sensitizedmice challenged with the same antigen developacute BHR, increased IgE levels, infiltration ofeosinophils and mucus overproduction, whichmimics bronchial asthma in man. The role of Th2

type cytokines for the development of BHR is

R. Moser et al. / Toxicology 158 (2001) 75–8380

established. Recent data suggest that beside IL-4and IL-5, IL-13 plays an essential role in BHR(reviewed in Wills-Karp, 1999). Therefore, inboth dermal contact and respiratory hypersensi-tivity reactions Th cells play a central initiatingrole. While the recognition is class I- and II-re-stricted and mediated by T cells, a role of CD1-restricted T cells in allergic reactions has beensuggested (Spinozzi et al., 1998). This model canbe applied to any antigen or hapten to test forpotential respiratory hyperreactivity and a shortoutline of the experimental protocol is as fol-lows: mice are first immunized with the antigen(or hapten coupled to protein) in alum (twoweekly injections) followed by respiratory chal-lenge (intratracheal or nasal application of anti-gen or aerosol challenge.) A typical response toovalbumin in mice sensitized to ovalbumin com-prises of accumulation of mononuclear cells andeosinophils in peribronchial tissues and mucushypersecretion. Sensitized mice challenged withsaline instead of ovalbumin do not develop anymicroscopic signs.

7. Non-cytokine factors involved in allergicresponses

Beside cytokines and chemokines, other fac-tors play a role in allergic responses. Transgenicmice with inactivated IgE, CD23, FcoR, lipooxy-genase, cyclooxygenase, stem cell factor (SCF),might be useful in further investigations. In ad-dition, mice that lack mast cells (w/wv), NKcells, T cells, B cells or any other effector cellsof the immune system could be used.

8. Conclusion

Transgenic mouse models are potentially use-ful models to unravel the pathophysiologicalmechanisms of drug hypersensitivity. As cytoki-nes are key regulators of immune responses, theuse of mice with overexpression or deletion ofcytokine genes are likely to be useful.

Recent highlights were obtained from IL-4RaKO and IL-13 KO mice demonstrating the criti-

cal role of IL-13 in the development of respira-tory hyperreactivity (Wills-Karp et al., 1998).Mice overexpressing Th2 cytokines, such as IL-4,IL-5 and IL-13, are likely to be of interest todesign sensitive models of allergic reactions. Be-cause the development of allergic/hypersensitivityreactions is modulated by genetic predispositionin mice as in man, allergens cause reactions inonly a fraction of exposed individuals. There-fore, the development of predictive models ofhypersensitivity is difficult as previously dis-cussed (Choquet-Kastylevsky and Descotes,1998).

In conclusion, animal models only partly mir-ror the reactions seen in man. Extensive investi-gations are therefore required prior to makingany recommendations on the potential predictivevalue of murine models in man.

Acknowledgements

This work was supported by MRC grant 41538 Harry Crossley Fund 43587 Cape Town andNRF Pretoria.

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