Autoimmune phenomena in chronic chagasic cardiopathy

6

Parasitology Today, vol. 1, no. I, 1985

Acknowledgements: The author is supportedby research grants from the United States Public Health Service A I 14848 and AI 17041.

determine reactivity against xenogeneic the hypothetical role of autoimmtmity in the heart antigens, possibly by cross-reaction. pathogenesis of Chagas disease, it does not On the other hand, other investigators have seem unreasonable to ask for definitive not been able to establish a correlation bet- proof. Such proof would have to be preween the presence of anti-heart reactivity ceded by clear evidence of cross-reactivity and the presence of heart disease in chagasic between the parasite and host antigens and patients, using a blastogenesis assay22. take the form of incontrovertible evidence When considering humoral or cell- that immunization with the cross-reacting mediated immunological reactivities bet- antigen(s), as weU as passive transfer of ween T. cruz/ and host tissue antigens, either antibodies or lymphocytes specific for it should be borne in mind that the immune this antigen, result in the production of system is 'programmed' to overlook self an- characteristic chagasic tissue lesions. Until further research definitively retigens. Thus, the mere sharing of epitopesif ever convincingly demonstrated - cannot solves the controversy surrounding autoimbe construed as reliable evidence that the re- munity in Chagas disease, it would be wise levant parasite epitope will elicit an immune to remain open-minded and to support and response in the host or, if it did, that it pursue all reasonable prospects towards the would be necessarily deleterious to the host. development of effective immunoAfter years of hearing and reading about prophylaxis against T. cruz/infection.

Autoimmune phenomena in chronic chagasic cardiopathy L. Hudson Depar~ent of Immunology St Georges Hospital Medical School London SW 17 ORE UK

'

In many instances the distinction between infection and disease might seem an unproductive semantic quibble that adds nothing to our understanding of the mechanisms of pathogenesis. However, this distinction serves to highlight the central paradox of South American trypanosomiasis and chronic chagasic cardiopathy: infection and disease are shifted in time and are not quantitatively related23. Very often, Chagas disease develops only many years after initial infection; its severity shows no direct correlation with the intensity of parasitisation and it attains its most severe expression at a time when the" parasite is virtually undetectable by all but the most sensitive multiplicative techniques (xenodiagnosis or haemoculture). Significantly, clinicians and pathologists have long known that the disease assumes its most extreme form in patients who mount the most vigorous immune response and so most effectively control their infection23. It seems, therefore, that for a full explanation of the pathogenesis of chagasic cardiopathy of digestive syndromes, the parasite alone is not enough. The earliest description of the histopathology of T. cruz/infection and Chagas disease, by Vianna in 1911, laid special emphasis on the observation that 'nests' of in(~)1985, Eisev,er Science Publishers B V Amsterdam 0169~475~85/¢~2 00

tracelluhr amastigotes failed to attract the attention of the immune response until after parasite transformation and host cell rup-' ture 24. In the chronic phase, histological examination frequently revealed a progressive inflammatory process leading to myocardial destruction and fibrosis in the complete absence of parasites. These observations were confirmed by Andrade25 in a careful study of the development of cardiac lesions in experimentally infected dogs. He noted that although inflammatory infiltrates were initially confined to parasitic foci, they disseminated later in the acute phase to involve uninfected muscle fibres. As these changes coincided with the resolution of parasitaemia under immune pressure, Andrade suggested that dissemination might result from the immune response recognising host cells modified by the passive adsorption of host antigensz6. Equally, however, this dissemination might also result from a change in the quality of the immune response, perhaps as a result of autoimmunity. Cross reactive antigens

By the criteria we apply to diseases with more familiar and accepted autoimmune associations (for example, systemic lupus erythematosus or Hashimoto's

Parasitology Today, vol. I, no. I, 1985

thyroiditis27) the e~istence of autoimmune cross reactivity in Chagas disease is not open to doubt. Several autoantibodies have been described either from naturally infected patients or experimentally infected animals (Table 1). Although these serum antibodies were elicited by T cruz/ infection, they could be specifically removed by absorption in uninfected host tissue 6. Further confirmation of the existence of cross reactive epitopes came from the application of monoclonal antibody techniques. Hybridomas were immortalised from the spleens of mice immunised with either dorsal root ganglia from uninfected rats 3 o r with T. cruz/epimastigotes 2s, and reciprocal screening identified two monoclonal antibodies which defuaed different epitopes shared by parasite, neurones and cardiac muscle. In the case of the former antibody, CE5, the relatively trivial explanation that these findings mighl: result from passively adsorbed host antigens was excluded by antibody-specific immunoprecipitation of an 87.5 kDa component from 35--S methionine labelled ~inv/tro translation products of poly(A) + ]LNA from CL strain epimastigotes,

Immunityand disease

7

Table I. Autoimmune serology of Chagasdisease (Cross reactivity between host and parasite has also been described using T-lymphocTte~ However, some of the experiments are difficult to reconcile with the known requirements for the expression of specific lymphocyte effector function. For full discus-. sion see ref. 40.) Antibody

Epltope expressed on

Comments

EVl

endocardiaI-vascularinterstitial tissue

Present in 90% of chronic carcliopathy casesand40% of indeterminate stage patients. Absorbed with T. cruz/epimastigotes and T. b. rhedesiense

anti-

Schwanncell sheath

Positively correlated with EVI. Absorbed out with T. cruz/epimastigotes

6

subpopulation of peripheral and central neurons

Staining reduced by absorption with T. cruzi and mouse liver membranes

36

IgM anti-lgG

Present in 95% patients with acute disease,25% with chronic disease

37

sarcoplasrnicreticulum adenosinetriphosphate

Present on skeletal and heart muscle

38

laminin in connective tissues

Also bindsstrongly to trypornastigotesand amastigotes but not to collagen,fibronectin, chondornectin or proteoglycan

39

peripheral

nerve antinerve bocly

antiimmunoglobulin

Reference 35

(rheumatoid

factor) antistriated muscle

antilaminin

The first direct a~tempt to uncover the potential role that cross reactive immunisadon might play in disease pathogenesis came from the experiments of Teixeira and mother using serum or lymphocytes, in the his colleagues29 who had previously made absence of parasites. Transfer of humoral an extensive study of the pathology of T. serum may confer resistance to infection, cruz/ infection in rabbits. These inves- but does not transfer pathology. Adoptive tigators immunised rabbits with dead para- transfer experiments require inbred animals sites each week for 9 months and found that and so, as the more usual models of chronic a high proportion of the rabbits developed cardiopathy (man, rabbit and dog) were pathology very similar to that resulting from excluded, Laguens et al. 30 transferred lymlive infection. Thus infection was not re- phocytes from infected mice with myocarquired to produce di,,~ase. dial infiltration and ECG abnormalities, to Regrettably, as noted by Kierszenbaum uninfected recipients. The recipient mice above, the interpretation of the initial exper- showed no infection but did develop ECG iments was clouded by the realisation that changes and histological evidence ofinfdtrathe immunogen had been prepared from don within the surprisingly short time of 4 parasites grown in host cells and so might days. have contained contaminating host compoAlthough these findings are in accord nents. While the fmcling was not in doubt, with disease transfer, it would be prudent to its genesis was. Although the same group treat this single report with caution until it is has since made simi]lar observations in in- confirmed using more stringent criteria. bred rabbits immunised with parasites The techniques for ECG measurements as grown under unimpeachable conditions, described 3° cannot be applied to mice with the force of the original observation has the same facility as they are applied to man. been lost. For example, only 3 ECG leads (bipolar The most convincing demonstration of leads I, II and III with augmented unipolar the role of the immune response in the leads aVR, aVL and aVF) can be placed with pathogenesis of chronic cardiopathy would sufficient reproducibility, and recordings be the transfer of disease from one subject to can only be made under general anaesthesia

Trypanosoma cruzi Epimastigote

>

8

Parasitology Today, vol. I, no. t, 1985

b L. Hudson

Fig. I Immunocytochemical labelling by CE5 monodonal antibody. (a) viable or formaldehydefixed amasttgotes ofT. cruzi show bright immunofluorescent staining, indicating that the epitope detected by CE5 is a surface determinant on the amastigates which is directly accessible to the immune system. In contrast, viable epimastlgotes showed no react/on with CE.5 until fixed end permeabilised by formaldehyde treatment, as shown here (b), Selective staining with immunoperoxidase shows that CE5 antibody also binds with Purkinje neurone cell bodies and dendrites in the rat cerebellar cortex (c). Purkinje neurons in the human cerebellar cortex show the some staining reaction.

Acknowledgements: While in no way associating them with the views expressed here, I am grateful to V, Britten and A.P. Johnstone for their helpful comments and to Kay Dorelli for typing the manuscript. Our research 'was supported by grants from the Medical Research Council and the Commission of the European CommuniZes.

with the attendant risk of an artefactual alteration in cardiac performance. Similarly, the fast heart rate of mice (449 _+ 65 beats/ min, mean + SD of 108 determinations: Hindmarsh, P.E., unpublished) compared to man (approx. 70 beats/min) means that very fast paper speeds, up to 250 mm/s, or electronic recording and analysis would have to be used to achieve sufficient discrimination. Fortunately these technical advances have now been made, and so one may confidently predict that these experiments in mice are likely to lead to the definitive model system in which to test the 'autoimmune hypothesis'. In the mould of classical autoimmune diseases, any immunologically mediated damage in Chagas disease would have to arise as a consequence of a secondary pathogenesis provoked by the initial parasite infection. This temporal relationship suggests that the disease ought to result from two separate but interrelated mechanisms, one parasitedependent and the other parasite-independent. There is no disagreement that the focal lesions of acute chagasic cardiopathy derive their character and distribution entirely from the numbers and disposition of the parasite within the myocardium. Thus, as parasitaemia resolves under immune pressure, the inflammatory infiltrate subsides and the lesions cease to progress. This is in direct contrast to the parasite-independence shown by chronic lesions. Barousse et al.31 observed patients with chronic Chagas disease who received immunosuppressive therapy for unrelated concomitant illness: although the removal of immune control resuited in a recrudescence of parasitaemia, chronic lesions were neither reactivated nor exacerbated - it seems the parasite was no longer important. forward Autoimmune phenomena in Chagas disease, thus far limited to autoantibodies and The way

cross reactive antigens, certainly exist. Additional circumstantial evidence combined with inconsistencies in the pattern of disease pathology compared to parasite burden, suggest that autoimmunity might be involved in chronic chagasic cardiopathy. Evidence from other diseases with well documented autoimmune components, for example insulin-dependent type I diabetes • 32 mellitus , suggests that the ability to produce autoantibodies might predispose to disease, but that the autoantibodies are n o t sufficient in themselves. This notion is reinforced by the fmding of autoantibodies in other parasitic diseases with no known autoimmune implications (for example, antilaminin antibodies in leishmaniasis33) and by the many inconclusive attempts to demonstrate a pathologic role for EVI autoantibodies in Chagas disease. It seems likely, therefore, that overt chronic disease might also require cell-mediated immunity30 or some unrecognised precipitating factor, such as a concomitant viral myocarditis, for its full expression. Experimentation in our own and other laboratories has moved inexorably towards model systems which should finally provide a definitive test of the supposed immunopathogenesis of Chagas disease precipitated by T. cruz/ infection. We have developed oa mouse model which permits sequential monitoring of ECG and plasma concentration of specific muscle enzymes after low level infection. Although it does n o t develop all aspects of each stage of the clinical disease, it provides a reproducible model of mild cardiopathy which is susceptible to proper experimental manipulation in statistically valid groups. The simultaneous development of T- and B-cell hybridomas and cloned lymphocyte lines suggests that we will have not only a means of uncovering the disease mechanism but also the tools with which to effect its immunological regulation in a manner analagous to that shown in experimental de° myelinating disease34. As long as the evidence for autoimmunity in chronic chagasic cardiopathy remains circumstantial, its study might seem unproductive and a possible delay in the progress towards immunoprophylaxis. However, it is important to remember that ethical considerations for proper safety testing do n o t demand complete proof but only reasonable suspicion. We have that reasonable suspicion. References

1 2

Cassio, P.M., et aL (1974) Circulat~ 49, 13-21 Santos-Buch, C.A. and Teixeira, A.R.L. (1974).

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3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

ft. Exp. Med. 140, 38-53 Wood, !.N. et al. (1982) Nature 296, 34-38 Vega, M. de la, Damilazto, G. and Diez, C. (1976) ft. Parasitol. 62, 129-130 Teixeira, A.R.L. etal.(1978)Am.ff. Trop. Med. Hyg. 27, 1097-1107 Khoury, E.L. et al. (197,)) Clin. Exp. lmmunol. 36,. 8-15 Torres, C.M. (1941) Mere. Inst. Oswaldo Cruz 36, 391--404 Muniz, J. and Azevedo, A.P. (1947) O Hospital 32, 165-183 Khoury, E.L. et al. (1978) Am. J. Clin. Pathol. 69, 62-65 Cassio, P.M. et al. (1974) Circulation 50, 1252-1259 Hiibsch, R.M., Sttlzer, ,~.J. and Kagan, I.G. (1976) J. Parasitol. 62, 523-527 Cassio, P.M. et al. (1977) Am. ft. Pathol. 86, 533-539 Bretana, A. and O'Daly, II.A. (1976) Int. ft. Parasitol. 6, 376-386 Szarfman, A. et al. (1975) Am. ff . Trop. Med. Hyg. 24, 19-24 Schmufiis, G.A. eta/. (1978) J. Infect. Dis. 138, 401--404 Khoury, E.L. et al. (]983) Clin. lmmunol, lmmunopathol. 27, 283-288 Khoury, E.L. and Fields, K.L. (1980) Lancet i, 1088 Hudson, L. (1983)J. Cell Biochem. 21,299-304 Hudson, L. (1983) in Cy~pathology of Parasitic Disease (Ciba Foundation Symrosium 99), pp. 234-251. Pitman Books, London, UK Wartenberg, J. and Brostoff, J. (1973)Br. HeartJourha/35, 845--848 Todd, C.W., Todd, N.R. and Guimaraes, A.C. (1983) Infect. Immun. 40, 832-835 Mosca, W. and Plaja, J. (:[981)J. Clin. Microbial. 14,

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1-5 Teixeira, A.R.L. (1979) Ball. WId Hlth Org. 57, 697-710 Vianna, G. (1911)Mere. Inst. Oswa/do Cruz 3,276-293 Andrade, Z.A. (1983) in Cytopathology of Parasitic Disease (Ciba Foundation Symposium 99), pp. 214-233. Pitman Books, London, UK Hudson, L. and Ribeiro dos Santos, R. (1982) in Immunology of Cardiovascular Disease. (ed. M.H. Lessof) pp. 141-155, Dekker, New York Waksman, B.H. (1984) Immuno/. Today 5, 346-348 Snary, D. et al. (1983) Clin. Exp. IrronunoL 54, 617-624 Teixeira, A.R.L., Teixeira, M.L. and Santos-Buch, C.A. (1975)Am.J. Pathol. 80, 163-178 Laguens, R.P. et al (1981) Medicina (Buenos Aires) 41, 40-43 Barousse, A.P. et al. (1980) Medicina (Buenos Aires) 40, 17726 Bottazzo, G.F., Florin-Christensen, A. and Doniach, D. (1974) Lancet ii, 1279-12832 Avila, J.L., Rajas, M. and Rieber, M. (1984) Infect. Inmnra. 43, 402-406 Ben-Nun, A., Wekerle, H. and Cohen, I.R. (1981) Nature 292, 60--61 Cassio, P.M. eta/. (1974) C/rcalatwn 49, 13 Ribeiro dos Santos, R. (1979) Tropemned. Parasit. 30, 19-23 Cabral, H.R.A. et al. (1966) Rev. Fac. C/enc. Med. Cordoba 24, 395-398 Sadigursky, M., Acosta, A.M. and Santos-Buch,C.A. (1982)Am.J. Trap. Med. Hyg. 31,934-941 Szarfman, A. et al. (1982) ft. Exp Med. 155, 1161-1171 Hudson, L. and Britten, V. (1985) Br. Med. Bull. 41

(in press)

Approximate world distribution of human Chagas disease