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7171717171Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I: 71-80, 1999

Immunopathology of Chagas DiseaseZilton A Andrade

Laboratório de Patologia Experimental, Centro de Pesquisas Gonçalo Moniz-Fiocruz, Rua Valdemar Falcão 121,40295-001 Salvador, BA, Brasil

The main clinical forms of Chagas disease (acute, indeterminate and chronic cardiac) present strongevidences for the participation of the immune system on pathogenesis. Although parasite multiplicationis evident during acute infection, the intense acute myocarditis of this phase exhibits clear ultrastruc-tural signs of cell-mediated immune damage, inflicted to parasitized and non-parasitized myocardiocytesand to the endothelium of myocardial capillaries (microangiopathy). Inflammation subsides almostcompletely when immunity decreases parasite load and suppressor factors modulate host reaction, butinflammation does not disappear when the disease enters the indeterminate phase. Inflammation be-comes mild and focal and undergoes cyclic changes leading to complete resolution. However, the pro-cess is maintained because the disappearance of old focal lesions is balanced by the upsurge of newones. This equilibrium allows for prolonged host survival in the absence of symptoms or signs of dis-ease. The chronic cardiac form is represented by a delayed-type, cell-mediated diffuse myocarditis, thatprobably ensues when the suppressive mechanisms, operative during the indeterminate phase, becomedefaulted. The mechanism responsible for the transition from the indeterminate to the cardiac form, ispoorly understood.

Key words: Trypanosoma cruzi - immunopathology - myocarditis

The major aspects of the pathogenesis ofChagas disease remain poorly understood. Somecrucial pathological findings strongly suggest theparticipation of immunological fators. Inflamma-tory lesions in the myocardium show no direct cor-relation with the presence of parasites, regardlessthe clinical stage of the disease. The acute myo-carditis that follows primary infection with Trypa-nosoma cruzi, spontaneously subsides, after a pe-riod of two-three months in most cases, but doesnot disappear. In its place a mild focal myocarditisremains and may last for years without signs ofcumulative damage. Suddenly or slowly, severe,diffuse, progressive and fatal chronic myocarditisensues after a prolonged period of silent infection,in a small proportion of cases (about 30%). Thiscomplex chain of events is unusual to be seen inother parasitic diseases. It has stimulated researchon immunology and immunopathology of Chagasdisease, but efforts are often hampered by lack ofan adequate experimental model. Almost any mam-mal can be infected with T. cruzi. Acute infection(positive parasitemia in peripheral blood) can in-variably be documented a week or more after in-oculation. In some hosts infection will eventually

Fax: +55-71-359-4292Received 9 June 1999Accepted 9 August 1999

evolve toward spontaneous cure. Others will re-main infected, without signs of disease (indeter-minate form), but, different from the situation inman, none will develop progressive chronic myo-carditis, with cardiomegaly, arrhytmias and signsof chronic cardiac failure following a prolongedtime of silent infection. The dog is an exception.Young dogs usually develop severe acute diseasewhen naturally or experimentally infected with T.cruzi (Andrade 1984). When acute manifestationsabate, a period of latent infection follows andmay last for life. Eventually, chronic myocarditis,accompanied by cardiomegaly, arrhytmias,dispnea, peripheral edema will appear, without anyevidence of rapid resumption of parasite multipli-cation (Anselmi et al. 1966, Laranja & Andrade1980). Unfortunately, immunological data arescanty for this model, although a good amount ofclinical and morphological data are avaliable.Most of the subject to be discussed in this chapterwill be based on experimental studies made in dogs.This report will also be limited to heart involve-ment in Chagas disease. Lesions in other organs,especially involving the digestive tract, which mayresult in megaesophagus and megacolon, have beenlittle explored as far as immunopathology is con-cerned.

ACUTE PHASE MYOCARDITIS

Initially, the diffuse and intense myocarditisassociated with acute Chagas disease was consid-ered directly related to parasite multiplication. The

7272727272 Immunopathology of Chagas Disease � Zilton A Andrade

tissue forms of T. cruzi multiplied withincardiomyocytes and caused their rupture, liberat-ing inflammatory mediators of several kinds. Thiswas sufficient to explain the whole picture, sinceparasitism was easily observed in tissue sections.This contrasted to the situation with the chroniccardiac form, when parasites are rarely demon-strated by the usual techniques or even by a com-bination of them. Therefore, the pathogenesis ofacute myocarditis was considered radically dif-ferent from that of the chronic cardiac form. How-ever, closer observations of acute cases revealedthat, although parasites were present, the intensityof the inflammatory process did not always corre-lated with the overall presence of tissue forms ofthe parasites. Furthermore, destruction of non-parasitized cardiomyocytes was found to be anoutstanding feature, and that seemed to require amore elaborated explanation.

Sequential examination of experimental mate-rial shows that the first changes related to T. cruziinfection appear around ruptured parasitized fibers.Presence of immuno-globulin and complement can

be demonstrated in these focal lesions by immun-ofluorescence, revealing the early participation ofhumoral immune factors in pathogenesis (Silva etal. 1985). Initially focal reactions are seen scat-tered distributed throughout the myocardium a fewdays after experimental inoculation. This is soonfollowed by diffuse and more intense inflamma-tion. When this happens, isolated necrosis of non-parasitized cardiomyocytes becomes more or lessprominent. From then on, signs of cell-mediatedimmune damage can be ultrastructurally disclosed(Andrade et al. 1994). There is close adhesion ofmononuclear cells (small and large granular lym-phocytes, probably NK-cells and also macroph-ages) to cardiomyocyte membrane, with fusion ofmembrane at focal points, accompanied by focaldisruption of T tubules, sarcoplasmic reticulum,myofibrils, mitochondria and even intercallateddisks (Fig. 1). Damage to the cardiocyte some-times is multifocal, and leads to lytic or coagula-tive necrosis of large segments of the car-diomyocytes or even of their entire length. Cellsin close proximity to the damaged ones usually

Fig. 1: acute Chagas myocarditis. A dissociated myofiber (My) appears undergoing coagulative necrosis in the middle of lympho-cytes (L) and macrophages (M). There are points of contact and fusion between the membranes of the leukocytes and the myocell.Fragmentation of scalariform disks is evident (arrows). Electron micrograph, X5,000.

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appear entirely preserved. Possible causes for thesechanges have been suggested: (a) immunologicalinjury after adsorption of T. cruzi antigens on non-parasitized cells (Ribeiro dos Santos & Hudson1980); (b) ischemic injury due to platelet aggrega-tion and obstruction of myocardial capillaries(Rossi et al. 1984, Tanowitz et al. 1990); (c) di-rect or antibody-mediated cytotoxic damage byinflammatory and immune effector cells, includ-ing lymphocytes, neutrophils, eosinophils, mac-rophages and mast cells (Lopes et al. 1977, Tafuriet al. 1983, Cabral 1988, Molina & Kierszenbuam1989, Rossi 1990).

Besides these destructive changes of myocytes,a microangiopathy is also observed. It consists ofmyocardial capillary involvement, following closecontact between lymphocytes or macrophages tothe endothelial cells (Andrade et al. 1994). In thecontact points there occur membrane fusion and in-creased pinocytosis, tumefaction, vacuolation andcytoplasmic disruption of endothelial cells, associ-ated or not with platelet aggregation and fibrinmicrothrombi (Fig. 2). The role of a vascular factor

in acute Chagas myocarditis has been emphasized,especially by investigators who observed plateletthrombi within myocardial capillaries of mice (Rossiet al. 1984, Tanowitz et al. 1990). However, theultrastructural signs of cytotoxic damage to themyocytes seen in the canine model far exceededthose indicative of ischemic injury (Andrade 1984).On the other hand, microangiopathy appears as animportant clue suggesting the participation ofcytokines in acute Chagas myocarditis. Similar vas-cular changes have been observed in patients dyingof the so-called “leaky syndrome” after being treatedwith large doses of interleukin-2 (Cotran et al. 1987).Experimentally, rats treated either with recombi-nant interleukin-2, interleukin-1 and tumor necro-sis factors also exhibited similar myocardialmicroangiopathy (Yi & Ulich 1992, Zhang et al.1993). Recent studies have demonstrated thatcytokines associated with CD4 response of the Th-1 type, can be enhanced by the administration ofIL-12, which results in the production of INF-γ, IL-2 and TNF-α, causing reduction in parasitemic lev-els of infected animals (Silva et al. 1995,

Fig. 2: microangiopathy in acute Chagas myocarditis. A myocardial capillary appears with its lumen occluded by fibrinous throm-bus (Ft), while others are dilated and exhibit pinocytosis of endothelial cells. Electron micrograph. X5,000


Abrahamsohn & Coffman 1996, Cardillo et at. 1996,Hunter et al. 1996). Probably this mechanism ofprotection, when overwhelming, would contributeto capillary and other myocardial changes duringthe course of acute Chagas disease.

IMMUNOLOGICAL MODULATION

There are two basic host reactions to T. cruzi,which are represented by focal and diffuse inflam-mation. Focal reaction is parasite-mediated. It oc-curs anywhere the parasite enters host cells andthen multiplies, causing cell-rupture. Focal heartlesions, observed in human tissue in which para-site could not be demonstrated in routine histologi-cal sections, revealed the presence of parasiteantigen by immunohistochemistry (Higuchi et al.1993) or its genomic segments by PCR technique(Jones et al. 1993), which is in keeping with theidea that focal inflammatory reactions are parasite-induced.

The heart may present both types of reactions.Diffuse inflammation is not directly parasite re-lated. It occurs during the acute and chronic car-diac forms, while mild focal inflammation is thehallmark of the indeterminate form of the disease.

All other organs exhibit only focal reactionsfollowing parasitized-cell rupture, regardless thetype of reaction present in the heart. This simpleobservation reveals that some important pathogenicfactors of chagasic myocarditis are related to theheart itself. Also, by observing the cardiac histo-pathology one can follow the changes suggestiveof immunological modulation during the severalclinical stages of Chagas disease. The completepicture regarding what happen when diffuse in-flammation of the acute phase subsides and turnsinto a mild focal myocarditis, is poorly understood.The same is true for the transition of focal myo-carditis of the indeterminate form toward the dif-fuse and fibrosing chronic myocarditis of the car-diac form. Of course immunity factors, both hu-moral and cellular, contribute to decreasing para-site load. When cytokines patterns were investi-gated, transition from the acute form to the inde-terminate form was identified in infected childrenas a CD4-Th1 response changing into a Th0 pat-tern, with expression of both interferon-gamma andIL-4 (Smudio et al. 1998). There are evidencesthat immunossupression plays a role in diminish-ing and modifying the character of the inflamma-tory reaction ellicited by T. cruzi (Tarleton 1998).Depressed delayed skin reaction to parasite anti-gens and in-vitro demonstration of inhibition of IL-2 lymphocyte production have been reported inmice. Immunossupresed individuals with latent T.cruzi infection, especially those affect by Aids,

are seen to present acute exacerbation of the infec-tion (Tarleton 1993, Rocha et al. 1994).

THE INDETERMINATE FORM

The indeterminate phase of Chagas disease isdefined as the prolonged clinically silent periodthat follows the phase of acute primary infectionwith T. cruzi. The individuals present serologicaland/or parasitological evidences of infection, butremain asymptomatic and do not exhibit electro-cardiographic signs of heart involvement, nor X-rays abnormalities of the digestive tract.

Microscopically, infected individuals at the in-determinate form of Chagas disease reveal mildfocal myocarditis when their hearts are examinedfollowing biopsies (Mady et al. 1984, Palacios-Pruet al. 1989) or autopsies, that latter material beingavailable after suicidal or accidental deaths (Lopeset al. 1975, 1981). However, longitudinal clinicalstudies have demonstrated that subjects present-ing the indeterminate form tend to remain asymp-tomatic for prolonged time, and thus prognosis canbe considered good. This also indicate that lesionsof mild focal myocarditis are not cumulative.

Recent ultrastructural studies have demon-strated that the inflammatory cells comprise lym-phocytes, plasma cells, macrophages, and a fewpolymorphonuclears and mast cells (Andrade et al.1997). The inflammatory cells are accumulated infocal areas of the interstitial tissue of the myocar-dium, and do not establish cell-to-cell contactwith myocardiocytes and do not invade or destroythem, as occurs during acute disease (Fig. 3). Insome of these focal lesions there are evidences ofextracellular matrix degradation (dissolution, frag-mentation, and thickening of both collagen andelastic fibers), plus signs indicative of apoptosis(nuclear condensation and fragmentation, forma-tion of apoptotic bodies) (Fig. 4). These arechanges indicative of a cyclic mechanism of evo-lution for the focal inflammatory lesions (Andradeet al. 1997). As new lesions appear, the older onesare gradually removed by degradation of excessextracellular matrix (fibrolysis), and by resorp-tion of inflammatory cells (apoptosis). Apoptosisof inflammatory cells is so common that can beconsidered non-specific, but in focal myocarditisof Chagas disease it occurred simultaneously inalmost all cells of one single focus (Fig. 5). Prob-ably, the formation of new lesions is balanced bythe removal of older ones, thus allowing for pro-longed host survival, almost without clinical mani-festations. Of course manifestations may appearin the case focal lesions happen to involve strate-gic structures of the heart, such as the conductingsystem or the intrinsic autonomic nervous system.


Transition to chronic cardiac form probably oc-curs through disappearance of, or interferencewith, suppressive factors. Therefore, both parasitesand heart tissue appear to be important to explaindiffuse and progressive inflammatory lesions of thecardiac form, since parasites outside the heart willcontinue to induce focal reactions only (Barbosa& Andrade 1984). The factors responsible for thetransition from the indeterminate to the chronic,progressive stage of Chagas disease remain to beelucidated. This transition, in which the suppres-sion of cell-mediated immune responses ceases tooccur, seems to be related to reactivation of theinflammation. The inflammatory reaction of thechronic phase of Chagas disease in humans hasbeen recently characterized as consisting mainlyof cytotoxic CD8+ T-lymphocytes (Tostes et al.1994). These cells were seen to express theGramzyme A cytotoxic factor (Reis et al. 1993).It has been now a consensus that CD8+ lympho-

cytes are the main T-cell type responsible for im-mune activation in chronic chagasic myocarditis(Kumar & Tarleton 1998). These cells are activated,through Class I MHC molecules, by macrophagescontaining remnants of T. cruzi. The absence of aCD4+ T-cell response in the presence of T. cruziantigens suggests that the presentation of these an-tigens through Class II MHC molecules is inhib-ited. However, other evidence suggests that thedepletion of CD4+ lymphocytes in the chronic phaseof Chagas disease is related to selective apoptosisof these cells (Lopes et al. 1995). In the mouse modelof chronic Chagasic cardiomyopathy, stimulation ofT cells results in apoptosis of CD4+ cells but not ofCD8+ cells (DosReis et al. 1995) Taken together,the morphologic and immunologic observations re-viewed above support the concept that the indeter-minate phase of Chagas disease represents a stateof host-parasite equilibrium rather than of progres-sive chronic myocardial damage.

Fig. 3: indeterminate Chagas disease in the dog. Inflammatory cells are present in interstitial tissue, but do not make membrane tomembrane contact with myocardiocytes. Some cells are large granular lymphocytes (NK cells). Others exhibit apoptosis (Ap).Normal myocardiocytes appear at the center of the picture. Electron micrograp. X3,000.


THE CHRONIC CARDIAC FORM

The phase of chronic, progressive myocarditisand congestive heart failure which are associatedwith myocardial hypertrophy, myocyte degenera-tion, severe interstitial fibrosis and thickening ofthe basement membranes characterize the patho-logical picture of the chronic cardiac form ofChagas disease. One can assume that this formoriginates from the indeterminate stage of latentinfection, in 30% of the cases in man, and muchless for the dog. Some important points usually passunnoticed. A progressive, destructive and repara-tive process appears in the heart, previously af-fected during a long time by mild diffuse myocardi-tis. Although rarely, parasites can also be found inseveral other organs always accompanied by focalinflammation, a situation entirely similar to thatseen in the heart during the indeterminate stage.Except for such focal and rare microscopic para-site-related changes, no other abnormalities areapparent outside of the heart during the chroniccardiac form, except those derived from chronicpassive congestion and thrombo-embolic phenom-ena. No evident modification, from the status ofthe indeterminate phase, occurs regarding para-

Fig. 4: heart of dog with indeterminated Chagas disease. Chromatic nuclear materials (DNA) belonging to four disintegratingcells, probably cardiomyocytes, show different degree of condensation and fragmentation (apoptosis). Electron micrograph, X5,000.

Fig. 5: positive apoptosis test of almost all inflammatory cellspresent in a focal myocardial reaction, in a dog with the inde-terminate form of Chagas disease. Apoptag kit, X150.


sitism. Also, there are no definite changes regard-ing serum antibodies. There have been some at-tempts to investigate the behavior of mononuclearcells in peripheral blood, with the result that somesuppressor cells can be differentially stimulatedwith anti-idiotypic antibodies (Ouaissi et al. 1988,Gazzineli et al. 1988). Also, lymphocytes directlyisolated from the heart of patients with either theindeterminate or the cardiac forms of Chagas dis-ease have exhibited different patterns of reactiv-ity when in the presence of a particular myocar-dial epitope (Cunha Neto et al. 1995).

Experimental reproduction of the transitionfrom the period of silent infection to chronic pro-gressive myocarditis has been achieved with thecanine model (Andrade et al. 1987). Animals withknown acute infection at early age, and with pro-longed silent period of infection, with no electro-cardiographic alterations, were treated with lowdoses of cyclophosphamide. This procedure hasbeen assumed to exacerbate cell-mediated lesions,probably by selectively destroying suppressor T-lymphocytes, their precursor cells, or other ele-ments in the host-immune supressor network (Turket al. 1972, Schwartz et al. 1978, Colley et al. 1979,Liew & Russel 1983).

In contrast to non-treated dogs, that remainedwith mild focal myocarditis, all treated animals

exhibited intense and diffuse myocarditis (Fig. 6).Destruction of isolated myocells was extensivethroughout the contractile myocardium and theconduction system. Mononuclear cells were seenat the periphery and in the interior of disintegrat-ing myocardiocytes. Tissue forms of T. cruzi werenot detected in histological section although ex-haustively searched. Parasitemia in peripheralblood was always negative.

The mechanism of cell-mediated myocardialdamage remains to be elucidated. The suggestionfor an auto-immune process has been frequentlypresented, but has not been proved (Kierszembaum1986). A strong support in favor of an auto-im-mune mechanism for Chagas myocarditis camefrom experiments with transplanted neonatal heartsinto the external ear of isogenic mice (Ribeiro dosSantos et al. 1992). While the transplants remainedviable for a long time, when into the ear of normalreceptors, they were rejected if the receptor waschronically infected with T. cruzi. Effector cellswere identified as CD4 lymphocytes. However,recent data from Tarleton et al. (1997) contradictthese findings. They failed to observe rejection oftransplanted neonatal hearts placed into chroni-cally infected mice. Severe inflammation occurredonly when T. cruzi was directly injected into thetransplant, showing that the parasite, and not an

Fig. 6: heart of a dog with prolonged silent Trypanosoma cruzi infection after being treated with low dose cyclophosphamide. Achronic diffuse myocarditis, with some degree of fibrosis is now apparent. H & E, X100.


auto-immune process, was responsible for the ef-fect observed in experiments with transplantedneonatal hearts.

The auto-immune theory is supported by thelack of correlation between parasitism and chronicmyocarditis. However, the great age variation ofthe patients at the onset of clinical manifestations,the lack of exacerbations and remissions, the ab-sence of typical auto-antibodies at high and con-stant levels, the lack of involvement of other or-gans, are not in keeping with an auto-immune pro-cess. Some investigators have documented theidentity of epitopes present in the parasite andmyocardium (Sadigursky et al. 1982). These cross-reacting molecules could attract the destructivepower of the sensitized immune system. Similarly,the same could occur if parasite antigens happento be expressed in the myocyte membrane, or beadsorbed onto it (Ribeiro dos Santos & Hudson1909). There are several other suggestions to ex-plain myocardial damage in T. cruzi infection, allof them in needing of further research. Some datarefer to the idiotype/anti-idiotype network. Whengenerating antibodies against anti-T. cruzi globu-lins, the host organism would be in effect produc-ing antibodies that would have the same configu-ration as that of the parasite. These antibodiescould therefore, either directly react with or acti-vate immune cells against receptors present in thecardiomyocyte membranes. One such cross react-ing antigen found in man was represented by anenzyme, acetyl-cholinesterase. The anti-idiotypeantibody against this epitope was found to be morefrequent in the sera of patients with the cardiacform of Chagas disease than in those of the inde-terminate form (Quaissi et al. 1988). Other differ-ences regarding anti-idiotypic antibodies have beenregistered for the latent and active chronic formsof the disease. These findings are not only inter-esting for the supporters of the theory of auto-im-munity, but points toward the existence of a pro-cess of modulation or repression/des-repression inChagas disease pathogenesis.

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