CHAPTER 15Animal Models for Autoimmune and Inflammatory Disease

INTRODUCTION

The concept of autoimmunity was introduced in 1900 by Paul Ehrlich, who postulatedthat it would represent a fatal type of immune response mediated by antibodies

directed against an animal’s own antigens (Ehrlich and Morgenroth, 1906). Thus, theeffector mechanisms used in host defense could lead to severe tissue damage if misdi-rected toward the host. Ehrlich and Morgenroth coined the term “horror autotoxicus” tosignify a mechanism for avoiding autoimmunization. In most cases, animals are tolerantof their own self antigens, but under certain pathologic situations immune responses toself tissues do occur that can lead to severe tissue damage. In man, most autoimmunediseases develop spontaneously; several units (e.g, UNIT 15.3) in this Chapter will describeanimal models of human autoimmune disease that also develop spontaneously. On theother hand, major advances in understanding the pathogenesis of autoimmune diseaseshave been made over the past 25 years by studying animal models that have been inducedby injection of self-tissue antigens (e.g., see UNITS 15.1 & 15.2). In general, development ofdisease under these conditions requires that the tissue antigens be injected into syngeneicanimals in an adjuvant mixture containing potent bacterial antigens.

Immune damage in autoimmune disease can be mediated by either antibodies or T cells.In diseases such as systemic lupus erythematosis, chronic IgG antibody productiondirected against self antigens results in immune-complex deposition in the tissues,especially small blood vessels. Such diseases are classified as systemic autoimmunediseases, as opposed to tissue- or organ-specific autoimmune diseases that affect singleorgans or tissues. The first animal model of spontaneous autoimmune disease, the NewZealand Black (NZB) mouse, was discovered in 1959. It was later shown that crossingNZB with another New Zealand strain, New Zealand White (NZW), produced mice(NZB×NZW F1) with all the immunopathological features of human lupus nephritis(Howie and Helyer, 1968). The characteristics of many of the known strains of mice thatdevelop spontaneous systemic autoimmunity (including the two just mentioned) aredescribed in Table A.1E.4. Studies over the past few years have uncovered the geneticbases for the development of autoimmunity in many of these strains. Induction of chronicgraft-versus-host disease (GVHD) by injection of immunocompetent cells from onemouse strain into an allogeneic recipient is also frequently accompanied by many of theclinical and pathologic manifestations of systemic autoimmunity. Methods for inductionand characterization of GVHD, including selection of the most appropriate recipient anddonor strains, are described in UNIT 4.3.

This chapter will primarily describe protocols for induction of organ-specific autoimmuneand inflammatory diseases. The methods most frequently used for induction of organ-specific autoimmune diseases involve injecting animals with the normal counterparts ofthe affected tissues. Surprisingly, several years before Ehrlich and Morgenroth proposedthe concept of autoimmunity, a natural experiment in humans clearly demonstrated thatinjection of tissue-specific antigens could result in autoimmune disease in the host’snormal tissue. Rabies vaccination was widely used following the initial success of Pasteurin 1885; however, reports of neurological complications arising from this treatment

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appeared within 3 years of the introduction of the vaccine, and numerous cases werereported in the literature around the turn of the century. Although these complicationswere initially attributed to the attenuated rabies virus in the vaccine, neurologicalcomplications were also found to be frequently associated with administration of thekilled virus vaccine, which began in 1919. Thus, it appeared likely that the complicationswere related to injection of the nervous tissue used for preparation of the vaccine:individuals receiving the vaccine developed antibodies that reacted with brain extracts.The next major advance in the development of animal models for organ-specific autoim-mune disease was the demonstration by Rivers and associates (Rivers and Schwentker,1935) in the 1930s that monkeys repeatedly injected with central nervous system (CNS)extracts developed neurological dysfunction. Pathological examination of injected mon-keys disclosed extensive areas of myelin destruction associated with perivascular infil-trates of mononuclear cells. In the model developed by Rivers, induction of experimentalallergic encephalomyelitis (EAE) in monkeys required repeated injections of CNS tissue;however, studies in the 1940s demonstrated that the disease could be induced by a singleinjection of brain antigen emulsified in adjuvant containing killed Mycobacterium tuber-culosis (Morgan, 1947; Kabat et al., 1947). This observation formed the basis for thecreation of other experimental models of organ-specific disease, all involving injectionof tissue-specific antigens in some form of adjuvant.

Multiple sclerosis (MS) is the commonest demyelinating disorder of the brain and spinalcord. The etiology of MS remains unknown, but immune-mediated destruction of myelinis thought to be a likely pathogenic mechanism of CNS damage in this disease. EAEproduced by injection of CNS antigens has served for the past 50 years as an excellentanimal model for studying the pathogenesis of MS (McFarlin, 1977). The CNS antigeninvolved was initially identified as myelin basic protein (MBP), and certain sequenceswere shown to be encephalitogenic for a given species but not for others. The majordifferences in encephalitogenic activities of MBP from various sources result from minordifferences in amino acid composition. Other components of myelin, particularly proteo-lipid protein (PLP), were later shown to be antigenic as well. Early experiments showedthat EAE could be passively transferred with T lymphocytes, but not by antibodies,defining EAE as the prototypic cell-mediated, organ-specific autoimmune disease. Al-though EAE can be induced in many species, the two most widely studied animal modelsof EAE are those produced in rats and mice. For many years, mice were regarded asrelatively resistant to induction of EAE; however, modification of some of the methodsused in other species and use of genetically susceptible mouse strains have facilitated theuse of mice for studying EAE. One advantage of the mouse model is that whereas EAEin rats is a monophasic illness, in certain mouse strains relapsing forms of the disease canbe produced that mimic in many respects the relapsing nature of MS in man. Detailedprocedures for inducing active EAE in mice and for transferring the disease to normalrecipients (passive EAE) are described in UNIT 15.1. Methods of inducing EAE in rats,including adoptive transfer (passive EAE) of the disease to normal rats, are described inUNIT 15.2. One critical requirement for the successful use of either methodology isavailability of a purified preparation of CNS antigens. UNIT 15.1 describes biochemicalprocedures for purification of bovine MBP and PLP, and UNIT 15.2 describes techniquesfor purification of guinea pig MBP. Table 15.1.1 provides a very complete and usefulguide to the specific encephalitogenic peptide sequences of both MBP and PLP that arecapable of inducing EAE in many inbred mouse strains.

In man, type I or insulin-dependent diabetes mellitus (IDDM) is an autoimmune diseasethat results from destruction of the insulin-producing β cells of the islets of Langerhansin the pancreas. The specific lesion of the disease is called insulitis and is manifest as aninfiltration of the islets with mononuclear cells, including both CD8+ and CD4+ T cells.

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Autoantibodies against islet cells are frequently observed in IDDM, but their role indisease pathogenesis remains poorly defined. T cells specific for a number of islet-cellantigens have also been documented in IDDM, but the nature of the islet-cell antigen(s)responsible for initiation of the disease also remains unknown. There are two major animalmodels, NOD (non-obese diabetic) mice and BB (BioBreeding) rats (see Tables A.1E.4and A.1H.1), for the study of spontaneous diabetes; both have many features in commonwith human IDDM. The BB rat strain originated from a commercial colony of Wistar ratsat the BioBreeding Laboratories in Ottawa, Canada. The animals spontaneously developa disease similar to IDDM, including insulitis and autoantibodies against islet cells (Crisaet al., 1992). A diabetes-resistant (DR) subline of BB rats developed by selective breedingis histocompatible to the diabetes-prone (DP) rats. DP rats have a pronounced lym-phopenia due to a lack of T cells. T cells that express the RT6 alloantigen are particularlylow in DP rats, and their absence may be crucial to the development of diabetes.Restoration of RT6+ T cells by infusion from DR rats prevented diabetes in DP rats,whereas depletion of the cells by an anti-RT6 antibody triggered diabetes in DR rats. Acomplete guide to the use of BB rats to study experimental IDDM is presented in UNIT

15.3. In addition, protocols are provided for the induction of disease in DR-BB rats bydepletion of RT6+ T cells and for the prevention of disease in DP-BB rats by transfer ofRT6+ T cells from DR-BB rats. One important parameter that must be considered beforeusing the BB rat model is that both susceptibility and incidence of disease can be markedlyinfluenced by the presence of viral infections in the animal colony. Successful use of theseanimals requires constant monitoring of the animal colony; a support protocol is includeddetailing serological analysis of serum samples for the presence of antibodies to manyviruses and bacteria. UNIT 15.12 describes another method for inducing diabetes in ratsfollowing the removal of regulatory T cells. Thymectomy and irradiation are used torender animals partially T cell deficient. Using the animal model described, rats that donot normally develop autoimmune disease spontaneously develop diabetes following thisprocedure.

Protocols involving the use of diabetogenic NOD mice are described in UNIT 15.9. Unlikethe BB rat model, NOD mice do not exhibit T cell lymphocytopenia, but rather the inverse.In addition, NOD mice develop IDDM under the influence of their physical environment,particularly diet and exposure to microbial pathogens. A protocol for maintaining NODmice under conditions permissive of full expression of their autoimmune potential isincluded in this unit, as are methods for diagnosing and partially quantitating the degreeof insulitis developing in such mice. The unit also contains a protocol for isolating T cellpancreatic infiltrates in autoimmune NOD mice for use in adoptive transfer studies or forestablishing autoreactive T cell lines. A cell isolation method is provided to preparepancreatic islet cells as a source of islet antigens. Finally, a protocol is included to guidethe investigator in the selection and use of transgenes in NOD mice.

Rheumatoid arthritis is the most common autoimmune disease in man. No animal modelcompletely resembles the human disease. Rheumatoid arthritis is a complex disease andinvolves antibodies including an IgM anti-IgG autoantibody called rheumatoid factor;some of the tissue damage in this disease is caused by depostion of immune complexes.It is very likely that T cells recognizing a joint antigen(s) also play important roles indisease pathogenesis by secreting cytokines that initiate local inflammation within thejoint and promote the recruitment of granulocytes and macrophages, leading to destruc-tion of the joint. MRL-lpr/lpr mice (Table A.1E.4) have a form of arthritis with certainpathologic features in common with human rheumatoid arthritis, and these mice producehigh levels of IgM and IgG rheumatoid factors. Several different experimental modelsthat have some features in common with human rheumatoid arthritis have been developedin mice and rats, including collagen-induced arthritis, adjuvant arthritis, and streptococcal

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cell wall arthritis. UNIT 15.4 describes protocols for induction of adjuvant arthritis in therat. It should be noted that, in contrast to many of the other protocols in the chapter,adjuvant arthritis is induced by oil-in-water emulsions that contain M. tuberculosis butdo not contain tissue-specific antigens. In certain rat strains, disease can even be inducedby certain oils in the absence of mycobacteria. Antibodies play no role in the pathogenesisof this disease, and it can be regarded as a model of purely T cell–mediated jointinflammation and destruction. The nature of the autoantigen recognized by the disease-initiating T cells in this model is completely unknown, although it has been proposed thatthe T cells may recognize mycobacterial or cartilage proteoglycans or mycobacterial ormammalian heat shock proteins. UNIT 15.5 describes protocols for the induction andassessment of collagen-induced arthritis (CIA) in both the rat and the mouse. Followingimmunization with heterologous collagen, animals develop a polyarthritis that in manyrespects resembles rheumatoid arthritis in humans. In contrast to adjuvant arthritis, bothcellular and humoral immune responses have been shown to play important roles in thepathogenesis of CIA. UNIT 15.5 also contains detailed protocols for the preparation andpurification of native type II collagen from chicken tissues, as commercial sources of thiscritical reagent are scarce. UNIT 15.10 describes another animal model for arthritis in rats,which uses peptodoglycan-polysaccharide polymers (PG-PS) derived from the cell wallsof Streptococcus pyogenes. This model induces inflammatory responses with many of thefeatures that characterize human rheumatoid arthritis. In addition to arthritis, injection ofPG-PS can also induce hepatic, splenic, and peritoneal granulomas. Such responses areconsidered representative of several naturally occurring inflammatory disorders. UNIT 15.6

contains protocols for the induction of experimental autoimmune uveitis (EAU) in rodentmodel systems. This autoimmune disease in animals represents an excellent model forautoimmune inflammatory diseases of the posterior uvea in humans. Table 15.6.2 lists indetail the retinal antigens and peptides derived from them that can be used to induce EAUin Lewis rats. In the mouse, EAU can only readily be induced by immunization withpeptide 161 to 180 of human interphotoreceptor retinoid-binding protein. Similarly,induction of experimental autoimmune thyroiditis (EAT), an excellent model for autoim-mune (Hashimoto’s) thyroiditis in humans, by immunization with mouse thyroglobin, ispresented in UNIT 15.7.

Mouse and rat models for human myasthenia gravis, a T cell–dependent, antibody-medi-ated autoimmune disease, are presented in UNIT 15.8. This unit includes protocols forextraction and purification of Torpedo californica electric organ acetylcholine receptors(AChR), which are used as the immunogen to induce disease. In addition, detailedprotocols for measuring anti-AChR antibody, clinical disease progression, and the effectsof acetylcholinesterase inhibitors to reverse the disease are described.

UNIT 15.11 presents methods for inducing a form of immune complex glomerulonephritisknown as IgA nephropathy in mice and rats. This disease is distinguished from otherforms of immune-complex glomerulonephritis by virtue of the predominance of IgA asthe major class of Ig within the aggregates. Protocols describe the induction of IgAnephropathy using one of several inciting antigens including inert proteins (e.g., ovalbu-min) and Sendai virus. Methods used to evaluate disease are also presented.

UNIT 15.13 describes an adoptive transfer approach for inducing inflammatory bowel disease(IBD) in SCID mice. Reconstitution of such mice with CD45RBhigh CD4+ cells fromBALB/c mice results in IBD with many of the gastrointestinal lesions and histologicalfeatures present in human disease.

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UNIT 15.14 describes a mouse and rat animal model of inflammatory heart disease initiatedby CD4+ T cells. Myosin-induced autoimmune myocarditis is a paradigm of the immune-mediated cardiac damage believed to play a role in the pathogenesis of certain postinfec-tious human cardiomyopathies.

UNIT 15.15 describes a method to provoke the appearance of immunologically mediatedglomerulonephritis by injecting rats (or mice of certain strains) with mercuric chloride(HgCl2). HgCl2 induces B cell polyclonal activation under the control of CD4+ TH2-likecells. Autoantibody responses develop (e.g., anti-DNA, anti-laminin, anti–type II colla-gen), predominantly of the IgG1 isotype, along with antibodies against haptens such astrinitrophenol. Protocols for assessing parameters that characterize the disease are in-cluded in the unit.

UNITS 15.16 & 15.17 provide methods for inducing autoimmune disease of the ovary in mice.The 3-day thymectomy model described in UNIT 15.16 also induces stomach-specificautoimmunity. In UNIT 15.17, injections of a zona pellucida ovary-specific glycopeptide,ZP3, induces histological evidence of ovarian inflammation as well as antibody to thezona pellucida and T cell responses to the peptide. Methods for disease evaluation andsemiquantitation of ovarian pathology are included in these units. UNIT 15.17 also includessurgical procedures for adult oophorectomy, neonatal ovariectomy, and implantation ofovarian grafts into mice without endogenous ovaries.

UNIT 15.18 presents methods for inducing and measuring allergic asthma in mice. Theassessment protocols included in this unit allow for the in vivo investigation of airwayinflammation and airway hyperresponsiveness—the hallmarks of bronchial asthma.Protocols for measuring allergen-induced T cell responses and IgE responses are alsoincluded to determine the quality and grade of sensitization associated with airwayhyperresponsiveness.

UNIT 15.19 describes an animal model which is one of several that have been used to facilitateour understanding of the immunopathogenesis of inflammatory bowel disease. The modelemploys the use of TNBS, which induces severe colonic inflammation when administeredintrarectally in the SJL/J mice. The colitis that results from this procedures presentsclinical and histopathological findings that resemble those seen in Crohn’s disease. Theunit describes the critical parameters needed for successful induction of TNBS-colitis, aswell as methods for monitoring and grading disease levels. A support protocol for isolatinglamina propria mononuclear cells from mouse colons is also included.

Systemic lupus erythematosis is a systemic inflammatory autoimmune disease thatinvolves multiple organs. One of the major manifestations of this disease is the presenceof autoantibodies to self antigens. These antibodies form part of the immune complexesthat are responsible for much of the tissue damage seen during the course of this disease.UNIT 15.20 describes a series of assays that have been developed for the quantitation of theseautoantibodies in serveral different mouse models of systemic lupus.

LITERATURE CITED

Crisa, L., Mordes, J.P., and Rossini, A.A. 1992. Autoimmune diabetes mellitus in the BB rat. Diabetes Metab.Rev. 8:9-37.

Ehrlich, P. and Morgenroth, J. 1906. Studies on hemolysins. Fifth communication. In Collected Studies onImmunity (P. Ehrlich, ed.) pp. 71-87. John Wiley & Sons, New York.

Howie, J.B. and Helyer, B.J. 1968. The immunology and pathology of NZB mice. Adv. Immunol. 9:215-263.

Kabat, E.A., Wolf, A., and Bezer, A.E. 1947. The rapid production of acute disseminated encephalomyelitisin Rhesus monkeys by injection of heterologous and homologous brain tissue with adjuvants. J. Exp. Med.85:117-129.

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McFarlin, D.E. 1977. Immunologically induced demyelination. Excerpta Med. Int. Congr. Ser. 434:60-66.

Morgan, I.M. 1947. Allergic encephalomyelitis in monkeys in response to injection of normal monkey tissue.J. Exp. Med. 85:131-140.

Rivers, T.M. and Schwentker, F.F. 1935. Encephalomyelitis accompanied by myelin destruction experimen-tally produced in monkeys. J.Exp. Med. 61:689-700.

Ethan M. Shevach

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