Workshop IV. Aetiopathogenetic factors in Reiter's ... · Ann. rheum. Dis. (1979), 38, Supplement p. 102 WorkshopIV. Aetiopathogenetic factors in Reiter's syndrome Adjuvantdisease:

Ann. rheum. Dis. (1979), 38, Supplement p. 102

Workshop IV. Aetiopathogenetic factors in Reiter'ssyndrome

Adjuvant disease: pathology and immune reactivity

CARL M. PEARSON AND Y. H. CHANG

From the University of California, Los Angeles, California, USA

Adjuvant disease, or adjuvant arthritis, is a disorderthat is uniquely inducible in rats. After an injectionof Freund's complete adjuvant, or its incorporationin the oily base of some subcomponents of variousbacterial cell walls, a peripheral arthritis (Fig. 1)appears in about 10 to 14 days. This is sometimesassociated with urethritis in male animals and alsowith other lesions which include an iritis, perivascularlesions of the external ears and sometimes elsewhereon the skin, and, occasionally, internal lesions suchas small granulomas in the liver, etc. In addition anumber of animals develop a significant arthritis andperiarthritis in the caudal, or tail, joints and aboutthe vertebrae, especially near the intervertebral discs,the lumbar vertebrae, and also the sacroiliac joints.To the surprise of one of us,245b when trying to

induce an experimental myositis in rats by injecting

Fig. 1 Adjuvant arthritis.

into the skin between their scapulae a skeletal musclehomogenate combined with Freund's adjuvant,most of the rats developed a peripheral polyarthritisafter 10 to 14 days. This often affected all fourextremities, including many of the small digital joints,the wrists, and the ankles but rarely the moreproximal extremity joints. The tail seemed to beinvolved as did some of the lumbar vertebrae.

Shortly after this observation it was realised thatthis constellation of symptoms somewhat resembledone or more of the rheumatic diseases that afflicthumans, notably Reiter's syndrome (RS), psoriasiswith arthritis, rheumatoid arthritis, rheumatoidspondylitis, gonococcal arthropathy, arthritis andulcerative colitis, erythema nodosum, and irido-cyclitis.Of major importance at that time was the observa-

tion that plain Freund's adjuvant without the addedtissue homogenate could initiate the identicalsyndrome. Dr. Brian Newbould, who was at thattime working in our laboratory, then discovered thateven as little as 50 ,.g adjuvant induced the disease ina slightly shorter period of time when injecteddirectlyintooneortwo inguinallymphnodes. 234a, 234bThis amount of adjuvant was about 100 times lessthan the initial injection that had been given eitherinto the skin of the back, into one footpad, or into thebase of the tail-methods still used today.A large number of studies were done on this

model341 to try to find out why this disease developsin rats. Moreover, the model has become a routinescreening test for various anti-inflammatory com-pounds in laboratories throughout the world.

Clinical features

About 10 to 12 days after injection of adjuvant anarthritis and periarthritis suddenly appear in the

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IV. Aetiopathogenetic features Suppl. p. 103

joints of the extremities. These inflammatory lesions,especially in the distal small joints of the paws(Fig. 1), are characterised by redness, swelling ofvarying severity, and pain. The arthritis is oftenpara-articular and sometimes migratory. In rarecases it almost wholly immobilises the animal,although the proximal limb joints are seldomnoticeably affected. The intensity of the lesions maywax and wane for several weeks. Sometimes completeremission is followed by spontaneous recurrences,usually on or about the 60th and 100th postinocula-tion days.Death rarely occurs except in maximally affected

animals. The arthritis and periarthritis are oftenaccompanied by: (1) Reddish nodules in the externalears along the course of certain vessels. Thesebasically are the result of perivascular inflammatoryand oedematous reactions with secondary vascularcongestion. (2) A skin rash (Fig. 2), sometimesdiffuse, sometimes not very obvious, and best seen bycarefully shaving the skin of the back or flanks of theanimals. (3) An anterior uveitis (Fig. 3), whichoccurred in about 20% of at least two strains of ratsthat were carefully studied ophthalmologically andpathologically.320a (4) A urethritis and balanitis(Fig. 4) and prostatititis (Fig. 5) in some maleanimals.245b

Histological features

The histological features of the joint and periarticularlesions as well as the lesions in the eye, skin, genitals,and, occasionally, elsewhere have been the subject of

Fig. 2 Focal lesion in dermis of skin from the flankofanimal with adjuvant disease and diffuse dermatitis.The appearance of this lesion, called an 'onion peellesion', resembles that ofhuman psoriasis.

Fig. 3 Acute uveitis or iritis inanterior chamber with manyinflammatory cells and otherfeatures characteristic of iritis.Note retina in upper segment ofphotograph.

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Suppl. p. 104 Annals of the Rheumatic Diseases

osteogenesis (Fig. 6). Hence, in rapid fashion in themore severe cases, within a matter of 20 to 30 daysafter the clinical onset of the disease the small pawjoints may be invaded, destroyed, and fused by

Fig. 4 Intense balanitis and urethritis in male rat 16days after adjuvant injection. Material exuding fromurethra was sterile to culture by usual bacteriologicalmeans. Note also intense inflammation, deformity,and swelling of hind paws.

considerable study.46a, 171a, 245e The dominant earlyjoint lesion is oedema and sparse mononuclear(chiefly lymphocyte) inflammatory infiltration.Within two or three days the inflammatory responsebecomes intense. There is synovial proliferation,fibrin deposition, joint effusion, and profoundproliferation of the connective tissue elements,including fibroblasts and osteoblasts adjacant to a

peripheral joint (Fig. 6) and to an intervertebral discof the tail vertebrae (Fig. 7).The osteoblastic reaction causes a periosteal

Fig. 6 Intense periosteal osteogenesis with new boneformation as well as cartilage formation adjacent tobone and intense inflammatory reaction with fibroblasticreaction. Photograph taken adjacent to a metatarsaljoint.

Fig. 5 Histopathology ofprostateand seminal vesicules. Noteintense inflammatory infiltratemade up ofmononuclear cells,primarily lymphocytes with a fewplasma cells, and intensefibroblastic proliferative reaction.

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fibrous and bony bridges. In less severe casessynovial proliferation, some subchondral erosions,and periosteal new bone formation may cause onlyminor or moderate limitation of joint function. Thelumbar spine and tail (or caudal) vertebrae and

Fig. 7 Intense inflammatory infiltration adjacent tointervertebral disc separating two tail vertebrae. On theadjacent vertebrae was also significant new boneformation or periosteogenesis.

adjacent tissues may be affected in severe cases,leading to significant ankylosis (Fig. 8) with pro-found bony bridging (Fig. 9). Sometimes the lumbarspine is also involved, but not usually the thoracic orcervical spines. These features can readily be seenby x-ray examination.3l4a

Immunogenesis

After this syndrome was first described manyinvestigators tried to (1) define the mechanisms thatproduced the disease, especially during the latencyperiod; (2) ascertain the role of lymphocytes andlymph nodes in the genesis of the disease, and alsothe role of circulating antibodies; (3) vary theincidence and severity of the disease by experimentalmanipulations, including its complete inhibition; and(4) provide a model of inflammatory arthritis in thesearch for therapeutically effective anti-inflammatorydrugs.When the disease was originally observed it was

thought likely that a latent infection of some sortwas being activated in the rat. As the results ofother studies accumulated, however, an infection ofthe usual variety seemed less likely. This was becauEe(1) a bacterium or mycoplasma could never con-sistently or even occasionally be cultured from theaffected tissues285a; (2) penicillin and broad-spectrumantibiotics were ineffectual against the disease; (3)a 'germ-free' group of animals easily developed theentire disease syndrome245; (4) an inability totransfer the disease by contact between affected andhealthy rats or by inoculation of serum or affectedarticular tissues into normal controls; (5) the diseasewas transferred to highly inbred controls byinoculating them with viable sensitised lymphocytestaken from the lymph nodes and spleen or thethoracic duct of previously inoculated animals; and(6) this transfer failed if the lymphocytes werepreviously disrupted by repeated freezing andthawing.245f, 320c, 323a

Fig. 8 Ankylosed tail in affectedanimal secondary to pathologicalreactions described in the previousfigures.

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Fig. 9 Dissection of entire spineof rat showing profound bonyfusion of tail and lumbar vertebraebut sparing most thoracic and allcervical vertebrae. This type ofpicture was common.

On the other hand, some workers showed that avirus might be activated and, in turn, provoke animmune response, giving rise to arthritis and otherlesions.60c, 173b. The interferon-inducing substanceTilorone or purified interferons in small doses thatdo not possess anti-inflammatory reactions (theinterferon was induced in a tissue culture of fibro-blasts by infection with Sindbis virus) depress orcompletely inhibit the appearance of adjuvantdisease, especially when the Tilorone or interferon isgiven one or two days before and one or two daysafter the injection of adjuvant.

SENSITISING ANTIGEN

The precise sensitising 'antigen' has not beenidentified. It may be some component of the acid-fastbacillus (a prime component of Freund's adjuvant)such as one ofthe wax fractions, which in almost pureform, free of all detectable protein includingtuberculoprotein, can readily induce the disease.Several smaller protein molecules that have beenprepared from various other bacterial cell walls canalso induce adjuvant disease when injected in an oilyemulsion. These other bacterial cell wall preparationsinclude, among others, those obtained fromNocardia, Streptomyces, Streptococcus, andLactobacillus. Moreover, other studies have shownthat relatively small molecules such as peptidoglycans(comprised of a disaccharide and a hexapeptide) inmineral oil may induce adjuvant disease.l86a-f, 297aThe possible role of peptidoglycans from varioushuman sources or from bacterial cell walls in theinduction of certain human diseases such asrheumatoid arthritis, RS, and other humaninflammatory joint diseases as well as adjuvant

disease have been speculated upon extensively byHadler.143b

In a somewhat parallel series of investigationsSchwab278a showed that a polyarthritis can beproduced in rats by intraperitoneal inoculation ofwater soluble peptidoglycans obtained from the cellwall of group A streptococci. He showed, on the onehand, that the disease was chronic and, on the otherhand, that the peptidoglycans were generally non-degradable, that they were transported andsequestered in macrophages, the persistence of the(possibly antigenic) initiating stimulus being due toan inability or inefficiency ofmacrophages to degradethese bacterial cell wall constituents. He furtherspeculated that the defect in the handling of bacterialcell walls, observed in certain strains of animals,could be related to an immune deficiency.As had been found by many workers before,186a,exf

Schwab278a described the basic structure of thesewater-soluble preparations as oligosaccharides con-sisting of a backbone of N-acetylglucosamine andN-acetylmuramic acid which were associated withseveral peptide side chains that include a variety ofamino-acids, among them alanine, glutamine, andlysine.There has as yet been no proof that a virus is

involved in the syndrome. Only circumstantialevidence has been deduced, including the above-mentioned inhibition by Tilorone or interferon orother interferon-inducing agents. Furthermore, if avirus is involved it may be indirectly via theimmunological system.The theory that a hypersensitivity immune

reaction is at least partially responsible for adjuvantdisease, and also for certain human disorders such as

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RS, is based upon the following: (1) The results ofthe lymphocyte transfer experiment mentionedearlier. (2)Thatdevelopmentof the disease isinhibitedwhen all the lymph nodes that drain the site ofinoculation are resected.74a (3) That the latencybetween the time of inoculation and the onset ofdisease is invariably at least 10 days. (4) That arefractory state similar to immune tolerance isinduced when the animals are exposed early in life toadjuvant, or even when more than 100 Vg of deadtubercle bacilli are given in suspension during theincubation period after adjuvant inoculation. (5)The suppressive effect of whole-body irradiationwhen given immediately before adjuvant inoculation.(6) That the disease is suppressed by simultaneousadministration of heterologous antilymphocyteglobulin.74b (7) That the arthritis and other lesionsare accentuated by tuberculin (PPD) in doses of4 to 100 ,g given during the incubation period.74a(8) The observation that the arthritis can be trans-ferred only to native inbred animals by sensitisedlymphocytes between the seventh and eleventh post-inoculation days, when these cells must be competent(earlier they appear to lack full competence andhence need a lymph node environment in which todevelop further). (9) That the disease is producedwith greater regularity and more severely whensmaller amounts of adjuvant are injected directlyinto one or more of the inguinal lymph nodes.

A UTOIMM UNITYAn alternative explanation is that adjuvant diseasemay be an autoimmune phenomenon resulting inautoantibodies or specific sensitised lymphocytes, orboth, acting against the animal's own tissues.234a,245f, 320b There is so far no clearcut evidence ofautoimmunity in rats with adjuvant disease. Henceattempts to induce arthritis and other lesions in ratsby the intradermal inoculation of oil suspensions ofglycogen, silica, dried bovine cartiliage, extracts of avariety of other tissues, cardiolybin, and othermaterials has been totally unsuccessful.127a ButTrentham et al.314c have described the induction of asomewhat similar polyarthritis by type ;II collagenwhen placed in incomplete adjuvant. Other collagentypes did not induce any disease in similarly injectedrats. Possibly in this instance the type II collagencould be replacing the wax D, the tubercle bacillus,or the peptidoglycan and thereby be acting as acomplete adjuvant rather than conveying somethingspecific to the injected rats by type fl collagen.

ENDOGENOUS IMMUNOGENWe have recently obtained evidence suggesting thatthe immunogen(s) responsible for adjuvant disease

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N,N-dioctadecyl-N' ,N'-bis(2-hydroxyethyl)propanediamine(CP-20,961 )

Fig. 10 Structuralformula for alkyldiamine.

is/are endogenous rather than something introducedin the Freund's complete adjuvant.60c We found thata low molecular weight (MW=714), apparentlynon-immunogenic synthetic compound (called analkyldiamine (Fig. 10)) induced classic chronicpolyarthritis in Lewis rats that was morphologicallyindistinguishable in both time of appearance andclinical presentation as well as pathology from theclassic adjuvant disease induced by Freund's completeadjuvant.

Various anti-inflammatory compounds such asphenylbutazone and cortisone, or its derivatives, caninhibit the inflammatory lesions of adjuvant diseaseif they are given two or three days before and duringthe expected time of appearance of the arthritis, etc.If they are given at the time of adjuvant injection andthen discontinued during the 'incubation' periodthey have no effect. On the other hand, the inter-feron-inducers such as Tilorone and poly I:C aremost effective when given before and during thefirst two or three days after the injection of adjuvantand/or alkyldiamine. They may be discontinued afterthat, but still prevent adjuvant disease.To try to sort out the role of virus, humoral

response, and cell-mediated immunity Chang6Ocdevised a technique whereby he could test bothhumoral and cell-mediated immune responses to astrain of cells (EL4) when injected intraperitoneally inLewis rats. By prelabelling these cells with 51Cr itwas possible to ascertain, by release of 51Cr fromthese cells, that either the alkyldiamine or Freund'sadjuvant was capable of enhancing both cell-mediated and humoral immune responses to theimmunogen (either peptidoglycan or alkyldiamine)when coadministered. These findings strongly suggestthat the immunogen responsible for the developmentof adjuvant disease is endogenous-for example, aconstituent of host tissue, a viral protein, or somecomplex ofthe two.

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Genetic influence

Beyond biochemical, immunological, and potentiallyinfectious mechanisms there may well be a majorgenetic influence in adjuvant arthritis. Indeed, anumber of studies have shown that there are clearstrain differences in the response of inbred strains ofrats to adjuvant injections. One study showed thatadjuvant arthritis was induced in 100% of the Lewisinbred strains, in 450 of Long Evans, and in10 8 % of the AVN strains of rats.34a The incidencein a hybrid of the Lewis and AVN strains was 55%.A back-cross of the latter F1 with the Lewis strainproduced a strain with a 70% incidence of adjuvantdisease, whereas a back-cross with the AVN strainproduced a 35% incidence.A somewhat similar study186d showed that the

Lewis strain was the most highly susceptible toadjuvant arthritis, whereas incidence in the Fisherstrain was about 30%. The Buffalo strain of rats,with an incidence of less than 10% was almostcompletely resistant. Not only was the arthriticresponse quite variable from one strain to anotherl73abut also, in the four inbred strains tested, there wasa widely variable release of a ConA-reactive lympho-

MOe4+2) Man (HL-A

cyte subpopulation that exhibited a low response toPHA and PWM.Because of the striking similarity between adjuvant

arthritis and RS or AS, our view is that adjuvantdisease is linked to the Ag-B or H-1 histocompati-bility system in rats as RS and AS are linked to thehistocompatibility gene HLA-B27. A similar linkagewas postulated in rats. The major histocompatibilitycomplex has recently been investigated in consider-able details in rats,l26a, 143a and it would appear toharbour, just as in mice, one or several immune-response (Lr) loci that define lymphocyte response(Fig. 11). However, there has not been a systematicsearch for an allele linked with adjuvant arthritis inthe rat.The mechanism(s) that underlie(s) the adju-

vanticity of either complete Freund's adjuvant or of apeptidoglycan component of the bacterial cell wall orof the aklyldiamine is at present unclear. These agentsmay aid the development of the immune response bya non-specific stimulation of plasma cell or reticulumcell proliferation; by activating antigen-processingmacrophages; by intensifying the traffic of lympho-cytes and local lymph nodes draining the immunisa-tion site; or by a combination of these mechanisms.

Rot (Ag-B,H-I)(Tentative)

MLR(LD-I)

I(AJ(St)-3)

LAI1)

IrMLR(LD-1)SDs

*SDw

Fig. 11 Structure of the major histocompatibility complex of mouse and man and a tentative model ofa similar geneticcomplex in the rat. Note similarity between the complexes in man and rat, especially between the Ir loci and two lociSDs and SDw, which control respectively the stronger and weaker histocompatibility antigens in both man and the rat(from Gill and Kunzl2oa).

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Conclusion

The induction of adjuvant disease either by completeFreund's adjuvant or an alkyldiamine compound orother stimulants in inbred rats seems to be a reason-able model of RS since, in this animal species, anarthritis, an iritis, a urethritis, and sometimes adermatitis can be produced. These are features incommon with human RS.The weight of available evidence favours the view

that immunological phenomena underly the genera-tion of adjuvant disease. The induction of thisexperimental disease by intradermal inoculation ofan oily preparation of an apparent non-immuno-genic, low molecular weight, synthetic alklyldiaminesuggests that the responsible immunogen isendogenous to the animal. Such a conclusion couldnot have been drawn specifically when completeFreund's adjuvant or peptidoglycans from bacterialcell wall sources were used.The possible role of a virus is indicated by the

finding that interferon, prepared in vivo or in vitro,effectively suppressed the development of adjuvantdisease without effecting the immune response.These findings suggest that the disease may haveresulted from an aberrant immune response to someendogenous material(s)-for example, a viral proteinor some complex of viral protein and host tissue.That the variable host responses, probablyimmunological, are genetically dictated is suggestedby the finding that some inbred rat strains are highlysusceptible to the disease whereas other strains arepartially or wholly resistant. This suggests theinfluence of the major histocompatibility system inthe rat (Ag-B or H-1 system), much the same asHLA-B27 is associated with RS in humans.

General discussion

DR. E. ALBERT: Have you done a segregation studywhere you cross this resistant strain with a susceptiblestrain and then look in the F2 hybrids for histo-compatibility antigen and resistance or susceptibility,which would be the classical linkage test?PROF. PEARSON: We have not done any histocompati-bility studies as yet. We did some cross-breeding ofthe Lewis strain and the Buffalo strain several yearsago and compared the severity of the arthritis and theincidence. The arthritis appears in about 50% of theF-I offspring, which again suggests the importanceof genetic factors in addition, perhaps, to otherfactors.DR. ALBERT: Susceptibility is dominant overresistance ?

PROF. PEARSON: Yes, apparently.DR. A. CALIN: Are there instances of spontaneousadjuvant disease in rats?PROF. PEARSON: I have never seen or heard of anyspontaneously occurring polyarthritis. However,Dr. Byron Waxman, while inducing experimentalallergic encephalomyelitis (EAE) in his animals,occasionally observed polyarthritis instead of EAE.I think it is a matter of the relative balance betweenthe amount of brain extract and the amount oftubercle bacilli that were placed in Fruend's adjuvant.I have noticed the same phenomena using muscleextract and adjuvant. Other researchers, wheninducing experimental allergic nephritis, haveoccasionally seen arthritis also when the amount ofrenal tissue antigen was small versus the amount ofstimulating microbacterial agent in the adjuvant.DR. J. C. WOODROW: Do you think it might be worthlooking at cross-reactivity between some of thesesynthetic determinants and the host tissues? Sharedspecificities would at least take into account thepossibility of impaired immune response in somestrains.PROF. PEARSON: Yes indeed. Impaired degradationmight also be important. A number of bacterial cellwalls and their metabolites are non-digestible in thehuman for lack of adequate enzyme systems.Until they are disposed of by macrophage ingestionthey may remain on the spot for a long time andcould act as the primary stimulating factor or as anadjuvant for something else that is responsible forthe induction of the disease. Similar phenomenamight be operative in some of the B27-associateddiseases.DR. D. A. BREWERTON: Have any of the injections beengiven into the urethra or the gut instead of into theskin or the lymph glands?PROF. PEARSON: No, because other sites (sub-cutaneous, intramuscular, or intraperitoneal) are notvery satisfactory. It seems to be necessary to inject inan area of potent lymphoid drainage. Indeed, onecan block the disease completely by resecting theregional (popliteal, inguinal, and periaortic) lymphnodes within five days after the paw injection. Butfrom day seven onwards resection of the nodes willnot alter the normal course of the disease.DR. G. w. csoNKA: How does adjuvant diseasecompare with mycoplasma-induced arthritis inrats? After all, mycoplasmas have no cell walls?PROF. PEARSON: I have not had too much experiencewith mycoplasma-induced arthritis in the rat. I haveseen some when Dr. John Ward was at theMassachusetts General Hospital. Those animalsdeveloped a transient polyarthritis. They do not

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Suppi. p. 110 Annals of the Rheumatic Diseases

develop any of these other features that I havedescribed. I believe that mycoplamsa arthritis is adirect synovial infection and probably is relatively ifnot totally unrelated to what we are seeing inadjuvant-induced arthritis.

PROF. B. AMOR: Using your new compound, did youfind any difference in the reactivity of various strainsof rats?PROF. PEARSON: We have only tested it in the Lewisstrain so far.

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Documents

Workshop IV. Aetiopathogenetic factors in Reiter's ... · Ann. rheum. Dis. (1979), 38, Supplement p. 102 WorkshopIV. Aetiopathogenetic factors in Reiter's syndrome Adjuvantdisease: