Serum cytokines in chronic Chagas ' disease

We studied the serum levels of IL-2, IFN-γ and TNF in different clinical forms of Chagas' disease and in patients clinically compensated and decompensated. Cytokines measured in 91 patients with the chronic form of the disease did not differ from those of 13 normal individuals, suggesting the absence of activation of the TH1 pattern of lymphocyte response. There were no statistical differences among the 17 patients in the indeterminate form of the disease, the patients presenting either early (n = 4) or well-developed signs of cardiomyopathy (n = 62), the digestive (n = 4) or the mixed (n = 4) forms of the disease. Serum TNF was undetectable and IFN-γ levels did not differ between clinical forms and severities of Chagas’ disease. However, we found IL-2 higher levels in the 25 non-controlled patients than in the 66 controlled individuals (p < 0,001). We suggest that IL-2 dosage may be useful as an indicator of the need for more aggressive procedures. Key-words: Cardiomyopathy. Clinical form. Severity. Cytokines. Chagas' disease. 285 Serum cytokines in chronic Chagas' disease 1. Department of Medicine, Divisions of Internal Medicine and 2. Clinical Pathology Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, São Paulo, SP, Brazil and 3. Department of Medicine, Division of Endocrinology Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil. This work was supported, in part, by grants from FAPESPand CNPq-Brasil. Address to: Dra Laura Sterian Ward. Med. Int./Semio. Clín. Med./FCM. Cidade Universitária Zeferino Vaz, 13081-970 Campinas, SP, Brasil. Telefax: 55 19 2894107. Email: Ward@TURING.UNICAMP.BR Recebido para publicação em 21/5/98. Revista da Sociedade Brasileira de Medicina Tropical 32(3):285-289, mai-jun, 1999. ARTIGO Resumo Estudamos os níveis séricos da IL-2, IFN-γ e do TNF-α de portadores da doença de Chagas em suas diferentes formas clínicas, compensados e descompensados. As citocinas medidas nos 91 pacientes com a forma crônica da doença não diferiram dos níveis de 13 indivíduos controle. Não houve diferença estatística entre os 17 portadores da forma indeterminada da doença e os portadores de cardiopatia insipiente (n = 4), de cardiopatia bem estabelecida (n = 62), da forma digestiva (n = 4) ou da forma mista (n = 4) da doença. Os níveis séricos de TNF foram indetectáveis e IFN-γ não diferiu nas diferentes formas clínicas ou com a severidade da doença. No entanto, encontramos níveis mais elevados de IL-2 nos 25 pacientes não-contrololados do que nos 66 pacientes bem compensados (p < 0,001). Sugerimos que a dosagem de IL-2 possa servir como indicadora da necessidade de terapêuticas mais agressivas nestes pacientes. Palavras-chaves: Miocardiopatia. Forma clínica. Severidade. Citocinas. Doença de Chagas. Laura S. Ward1, Maria E. Guariento1, Gilberto A. Fernandes2 e Rui M.B. Maciel3 Citocinas séricas na forma crônica da doenca de Chagas Chagas' disease is estimated to affect 18 million people in Brazil6. The disease is one of the most important causes of morbidity and mortality in a broad area ranging from Argentina to the South of the United States16 28 . C h a r a c t e r i s t i c a l l y, after a self-limited acute phase, the immune response controls parasitism yet fails to completely eradicate the protozoan parasite. It is interesting that in the chronic phase, Chagas' disease becomes clinically manifested in less than 60% of the infected individuals who develop the characteristic cardiac and/or digestive

Laura S. Ward 1 , Maria E. Guariento 1 , Gilberto A. Fernandes 2 e Rui M.B.Maciel 3 Citocinas séricas na forma crônica da doenca de Chagas Chagas' disease is estimated to affect 18 million people in Brazil 6 .The disease is one of the most important causes of morbidity and mortality in a broad area ranging from Argentina to the South of the United States 16 28 .C h a r a c t e r i s t i c a l l y, after a self-limited acute phase, the immune response controls parasitism yet fails to completely eradicate the protozoan parasite.It is interesting that in the chronic phase, Chagas' disease becomes clinically manifested in less than 60% of the infected individuals who develop the characteristic cardiac and/or digestive manifestations of the disease 17 .The heart is the organ most frequently affected, followed by the autonomic nervous system, the lesion of which leads to the digestive form of the disease 17 .The pathogeny of the disease is still unclear, but three main mechanisms have been proposed for it: the neurogenic, the microvascular and the immunological-autoimmunity theories 12 22 .The few existing studies of the acute human infection demonstrate an abnormal immunological response to the parasite 25 26 .Immunologic findings in the chronic phase are more difficult to interpret, particularly considering the different clinical forms of the disease.Aseries of manifestations of immune-suppression have been demonstrated in experimental animals and in human, but the mechanism by which they develop remains obscure 13 20 21 26 .Depressed responses were reported in patients with either the cardiac or the digestive form of Chagas' disease although parasite antigen-specific and/or mitogen-induced PBMC proliferative responses were reported to be normal 4 .Normal levels of NK cell activity and normal IL-2 activated cytotoxicity (LAK) were shown in both symptomatic and non-symptomatic cardiac patients 23 .On the other hand, NK cell activity was found to be enhanced by in vitro exposure of PBMC to T. cruzi antigen in all but symptomatic patients 3 .Increased HLA-DR expression by peripheral blood T-cells and a higher frequency of CD28T cells have been described in both symptomatic and indeterminate phase patients but neither IL-2 or IL-4 message in PBMC nor serum IL-2r levels were found to be augmented 4 7 8 9 .
Because of their well-known actions in the inflammatory response, some investigators have attempted to relate cytokines levels to the severity of diseases 10 21 24 27 .We and others also demonstrated that chagasic patients with d i fferent clinical forms of the disease have peripheral blood mononuclear cells, which respond to T. cru z i antigen with a cytotoxic response that is selectively up regulated by IL-12 2 3 .However, we found no difference in the increment caused by cytokine treatment among the different forms of Chagas' disease 2 .
In the present study, we have examined some important cytokines levels in patients of chronic Chagas' disease with different clinical forms and severity.

Controls.
The Medical Ethics Committee of the Hospital das Clínicas (FCM/UNICAMP) granted permission to study 20 healthy medical students who volunteered to provide normal parameters for the cytokine assays.After careful clinical examination, a blood sample was obtained from each subject for serum IL-2, IFNγ a n d T N Fα determinations.All individuals were observed for a period of four to eight weeks and when they were suspected of any immune or inflammatory disease, their data were eliminated from analysis of the results.At the end of the observation period, only six males and seven females were selected (median age 24 years, range 21 to 27 years) and denoted group N.
Patients.Ninety one Chagas' disease patients were sequentially selected among the individuals who routinely attend the outpatient clinic.After a careful clinical observation and a review of each patient medical records, the subjects were classified as (-) when they had few or no symptoms, i.e., when they did not present manifestations of Chagas' disease and/or of any concomitant pathology, and as (+) when they were symptomatic or in an advanced stage of heart disease (functional type IV by the criteria of the Committee of the American Cardiology Association 41 .Again we discarded all data from patients suspected of any other concurrent active immune or inflammatory disease.
According to their clinical manifestation of the disease, the patients were divided into: . G roup I (indeterminate fo r m ) : s e v e n t e e n patients (  .Group IV (digestive form): four patients (1 M, 3F) aged 38 to 62 years (X = 51 ± 13 years) with megaesophagus or megacolon confirmed by radiological examination but no sign of cardiac disease.One of these individuals also had a well-controlled hypertension.
. Group V (mixed form): four patients (4 F) aged 37 to 67 years (X = 54 ± 7 years) with the combined manifestations of groups III + IV.No patient was using amiodarone.One of these individuals presented signs of non-compensated heart disease at the time of blood collection.

M e t h o d s .
Blood sampling took place between 9:00 and 13:00h.The samples were immediately processed and stored at -20 o C until assayed.IL-2, γ-IFN and TNF-α were measured by solid-phase enzyme immunoassays employing the multiple antibody sandwich principle (Intertest-2 Human Interleukin-2, Intertest-γ Human IFN-γ and Predicta Tumor Necrosis Factor-α ELISA kits), obtained from Genzyme Diagnostics, Cambridge, MA, USA.IL-2 kit had a detection limit of 100pg/ml.IFN-γ kit also had a detection limit of 100pg/ml.TNF-α detection limit was 10pg/ml.
Statistical analysis.The results are expressed as the mean ± standard error.Since the data did not show a normal distribution, they were analyzed by the Kruskal-Wallis (H statistic) test, the χ 2 test or Wilcoxon's rank sum test (Z statistic), as appropriate.Statistical analysis that did not reach a level of significance of p < 0.05 were considered as non-significant (NS).For statistical purposes, undetectable cytokine levels were assigned an arbitrary level corresponding to the least detectable value of the method.

RESULTS
Normal individuals did not differ from C h a g a s ' disease patients in terms of age or gender (χ 2 = NS).There was also no difference among the patients grouped according to their clinical form (I to V) (χ 2 = NS).The results of both normal individuals and Chagas' disease patients are shown in Table 1.IL-2 levels ranged from 200 to 5120pg/dL, γ-IFN ranged from 42 to 190 pg/dL, and TNF was undetectable in all individuals.T h e r e was no significant difference in serum cytokine levels between normal individuals and chagasic patients.Analysis of the 91 patients as a whole showed no correlation among IL2, TNF and IFN-γ levels.Considering the forms of clinical presentation of the disease, we observed a tendency of IL-2 levels to decrease from group I to group V, while IFN levels were randomly distributed (H = NS for both IL-2 and IFN).Sixty-six patients did not have concomitant pathologies or were considered to be clinically compensated (-), whereas 25 not controlled were classified as (+).Their serum cytokine levels are presented in Table 2.There was no difference in the IFN-γ (Z = 0.74; p = 0.22).However, serum IL-2 levels were higher in the non-controlled patients (Z = 3.895, p < 0.001).

DISCUSSION
Like many other parasitic infections, T. cruzi induces alteration in the immunological system of the host to circumvent his defense mechanisms 10 .IL-2, IFN-γ and TNF-α probably contribute to the control of parasite growth by triggering phagocytic cell activation and inflammation 1 .There are also evidences that, in addition to mechanical damage related to the presence of the parasite, infection by T. cru z i induces proinflammatory cytokine production in the myocardium itself, which may further exacerbate the pathology 5 12 .
Serum cytokine quantification has seldom been reported in Chagas' disease.One major reason for this is the widespread involvement of cytokines in inflammatory responses that may impair the interpretation of serum cytokine levels.In the present investigation, we studied the serum levels of IL-2, IFN-γ and TNF in different clinical forms of Chagas' disease.The serum cytokine levels in patients with the chronic form of the disease did not differ from those of normal individuals, suggesting a lack of activation of the TH1 lymphocyte response.Since most of our patients presented the cardiac form of the disease, these results agree with recent experimental studies suggesting that a lower level of TH1 activity may play a role in cardiac involvement, whereas an enhanced TH-1-type response is related to protective immunity and decreased pathogenesis 14 18 .Serum TNF-α levels were undetectable in both chronic chagasic patients and normal individuals.Serum IFN-γ levels also did not differ among clinical forms and the stage of the disease.Variations have been reported between symptomatic versus indeterminate form and in patients with cardiac and indeterminate form.However, cytokine profile does not seem to differentiate the clinical presentations of the disease.
In contrast, serum IL-2 levels were significantly increased in patients with decompensated cardiac function.IL-2 is a glycoprotein of pleotropic eff e c t s including the activation and proliferation of T cells, natural killer cells and B cells.Suppression of the IL-2 gene with deficient serum IL-2 and IL-2 receptor expression, apparently mediated by suppressor macrophages, is a well documented phenomenon occurring during acute infection which possibly plays an important role in the evolution of the disease 19 28 .Nonetheless, its role in Chagas' disease is still poorly understood.It is intriguing to note that IL-2 levels increase strikingly in non-compensated individuals, even though their distribution pattern in the different forms of the disease suggest an inverse correlation with more aggressive forms of the disease (Table 1).
It is frequently difficult to appraise the clinical condition of Chagas' disease patients, because of their misleading autonomic symptoms adjoined by specific social and cultural characteristics.Our results suggest that IL-2 may be related to the severity of the disease.Its increased level in non-compensated patients may be useful a useful tool in the clinical evaluation of these patients as an indicator of the need for more aggressive or invasive procedures.

.
G roup III (card i o myo p a t hy ) : sixty-two patients (20 M, 42F) aged 34 to 71 years (X = 55 ± 8.5 years) with Chagas' cardiac disease.T h e s e patients had been using amiodarone to control their arrhythmia at the dose of Mo = 400mg/day for about 4 years (Mo = 4 years).At the time of blood collection, 24 of these patients presented decompensated heart disease with clear clinical manifestations (22 patients were functional type III, and two patients were classified as functional type IV).

Table 1 -
Serum thyroid hormones and cytokines levels of normal individuals (group N) and Chagas' disease patients divided into groups I (indeterminate form), II (early cardiac form), III (cardiomyopathy), IV (digestive form) and V (mixed form).Values are reported as Mean ± SEM.