Mem Inst Oswaldo Cruz, Rio de Janeiro, 96(2) February 2001
Detection of Circulant Tumor Necrosis Factor-?, Soluble Tumor Necrosis Factor p75 and Interferon-? in Brazilian Patients with Dengue Fever and Dengue Hemorrhagic Fever
Departamento de Virologia, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
IDepartamento de Patologia, Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife, PE, Brasil
IIDepartamento de Virologia, Laboratório Central, Fusam, Recife, PE, Brasil
Pro-inflammatory cytokines are believed to play an important role in the pathogenesis of dengue infection. This study reports cytokine levels in a total of 54 patients examined in Recife, State of Pernambuco, Brazil. Five out of eight patients who had hemorrhagic manifestations presented tumor necrosis factor-a (TNF-alpha) levels in sera which were statistically higher than those recorded for controls. In contrast, only one out of 16 patients with mild manifestations had elevated TNF-alpha levels. The levels of interleukin-6 (IL), IL-1beta tested in 24 samples and IL-12 in 30 samples were not significantly increased. Interferon-g was present in 10 out of 30 patients with dengue. The data support the concept that the increased level of TNF-alpha is related to the severity of the disease. Soluble TNF receptor p75 was found in most patients but it is unlikely to be related to severity since it was found with an equivalent frequency and levels in 15 patients with dengue fever and another 15 with dengue hemorrhagic fever.
Dengue has been claimed to be an immunopathological disease (Halstead 1980). Patients with dengue hemorrhagic fever (DHF) initially show typical systemic manifestations of dengue fever (DF) such as fever, retroorbital headache, severe myalgia and rash. Thereafter, they rapidly develop a more severe and life-threatening syndrome with thrombocytopenia, diffuse capillary leakage, hemoconcentration and hypotension, which may be followed by hemorrhage. In the most severe form of the disease, dengue shock syndrome (DSS), the pulse pressure narrows and circulatory collapse leading to shock occurs (Halstead 1990, PAHO 1994). DHF/DSS is most often associated with a secondary immunological reaction caused by a dengue virus serotype different from that originating the primary infection, in which the immune response is crossreactive but not protective (Pang 1987, Kliks et al. 1989).
Monocytes/macrophages are considered target cells for dengue infection (Halstead 1980, Anderson et al. 1997). These cells may act as antigen-presenting cells and secrete monokines, which participate in T cell activation and production of macrophage activating cytokines. During a secondary immune response, dengue virus activated mononuclear phagocytes are thought to release pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha) would act by feedback on these cells and also on vascular endothelial cells stimulating secretion of interleukin-1beta (IL) and IL-6 into circulation (Yang et al. 1995, Hober et al. 1996, 1998, Bethell et al. 1998). These three cytokines have similar functions and are able to induce an acute phase response which can result in fever, liberation of chemoattractant factors, altered vascular permeability (Dinarelo 1996) resembling features present in DHF/DSS and therefore suggesting their involvement in the pathological features of dengue infection.
Pro-inflammatory cytokines have been detected in patients from Asia and Central America (Hober et al. 1993, Kuno & Bailey 1994) and also in Brazilian patients (Kubelka et al. 1995, Pinto et al. 1999). The association of cytokine production and severity has been suggested.
Recent studies in different viral infections and inflammatory diseases provide evidence that soluble tumor necrosis factor receptors (sTNFRs) may modulate an excess of biological TNF-alpha activity (Van Zee et al. 1992, Dinarello 1996). Alternatively soluble receptors could stabilize TNF molecules acting thus as agonists (Leuwenberg et al. 1994). Indeed, Bethell et al. (1998) related sTNFRs levels with disease severity. Most Brazilian patients with exanthematic dengue fever show elevated sTNFRs levels even in the absence of hemorrhagic manifestations (Pinto et al. 1999).
In the present study, we investigated levels of TNF-alpha, IL-1-beta, IL-6, IL-12, IFN-gamma and TNF-Rp75 in sera from Brazilian patients with dengue attended in Recife, State of Pernambuco, Brazil. For TNF-alpha, IL-1-beta and IL-6 determinations we used sera from 16 individuals (two children) attended in 1997 who had DF with usual clinical manifestations (fever, headache, myalgia and rash) devoid of hemorrhagic manifestation. Five other cases also classified as DF presented hemorrhagic manifestations such as epixtasis, gingival or abdominal bleeding. Three adults had confirmed DHF, following WHO criteria (PAHO 1994), with thrombocytopenia (<100.000 platelets/mm3) and hemoconcentration. Fifteen apparently healthy individuals were used as controls.
For IL-12, IFN-gamma and TNF-Rp75 determinations, we used sera from 15 individuals attended in 1999 who had DF without hemorrhagic manifestations and an additional 15 individuals with hemorrhagic manifestations and confirmed DHF with thrombocytopenia and hemoconcentration.
Dengue infection was confirmed serologically in all patients by IgM positivity using a Mac ELISA test (Nogueira et al. 1992).
Serum samples were obtained during appointment and were stored in aliquots at -20°C until use in the different assays. Levels of TNF-alpha and sTNF-Rp75 in sera were determined using commercial ELISA kits according to the manufacturer's prescriptions. Predicta from Genzyme was used for TNF-alpha, IL-1-beta and IL-6. R&D Systems Quantikine was used for IFN-gamma and sTNF-Rp75 and High Sensitivity Quantikine for IL-12. The following sensitivity limits were achieved in standard curves: TNF-alpha, 18.75 pg/ml; IL-6, 28.125 pg/ml; IL-1beta, 4 pg/ml; IFN-gamma, 15.6 pg/ml; IL-12, 0.78 pg/ml sTNF-Rp75, 7.8 pg/ml.
Statistical analyses were performed by Student's t-Distribution (tn-1=8, a=0.025 = 2.262, tn-1=9, alpha=0.025 = 2.306 or tn-1=14, alpha=0.025 =2.145) calculating a referential limit value for positivity, according to the following formula: Average of values from control samples + [Standard Deviation of values from control samples X tn-1;alpha=0.025)]. Determinations above referential limit values were considered positive.
TNF-alpha was detected in 6 out of 24 (25%) patients above the referential limit value for positivity, 41 pg/ml (Figure). IL-6 and IL-1beta were not detected in any patient (referential limit values for positivity were respectively 207,04 and 16,3 pg/ml).
Soluble TNF-Rp75 was detected in 21 out of 30 patients (73% above the referential limit value for positivity, 2715,1 pg/ml, Figure). IL-12 was detected in 4 out of 30 (13% above 7,66 pg/ml) and IFN-g was detected in 10 out of 30 patients (33% above 58,03 pg/ml).
Using the Fisher exact test (a=0.05; one sided), patients with hemorrhagic manifestations or DF/DHF showed a significantly higher frequency of positivity for TNF-alpha when compared with DF patients devoid of hemorrhagic manifestations (P= 0.0069). The sTNF-Rp75 elevation in patients was highly significant (P=0.0002) but the frequency of this factor was not significantly different between the two forms of the disease (DF had 11 out of 15 positive sera and DHF had 10 out of 15).
Hober et al. (1993) have detected the pro-inflammatory cytokines TNF-alpha and IL-6 in Asiatic patients with DHF/DSS during Dengue-3 infection. Moreover, the follow up of a child infected by Dengue-4 demonstrated that an increase in severity was followed by elevation of serum levels of TNF-alpha, IL-6 and IL-1beta (Iyngkaran et al. 1995). The only study previously performed in Central America correlated higher cytokine levels with child hospitalization (Kuno & Bailey 1994) but no other severity parameter was described. We observed in an earlier work, one fatal case in Rio de Janeiro with a serum level of 900 pg/ml TNF-a (Kubelka et al. 1995). In our present study in Brazil, all three dengue patients with TNF-alpha levels higher than 100 pg/ml showed an elevated grade of severity, supporting the concept that this factor is produced in greater quantities during DHF or DF with hemorrhagic manifestations.
Soluble TNF-Rp75 was detected in the majority of patients tested but it was not possible to associate its presence with severity in contrast to the situation described by Bethell et al. (1998). In that work DHF patients had altered sTNF-Rp75 levels in sera but only patients with fatal shock had marked raise of the soluble receptor. We have only witnessed one case of recovery from shock; it may be the case that the association between elevated sTNFRp75 and severity is only true in very severe cases or even fatal ones. However, we should emphasize that most Brazilian patients are adults whereas the bulk of Asiatic cases involve children. Furthermore, genetic and geographical factors may influence this association.
High TNF-a levels have been more clearly correlated with hemorrhagic manifestations and/or confirmed DHF. TNF has many effects on the endothelium and stimulates the production of vasodilating substances such as prostaglandin I2 and nitric oxide. Also, TNF-a has a powerful antifibrinolytic action, by decreasing the production of plasminogen activators and increasing that of plasminogen activators inhibitor 1 (Vassali 1992, Mantovani et al. 1997). Nevertheless, investigations are far from elucidating the complex mechanisms of immunopathology during dengue infection; more cases in different countries and several immunological parameters remain to be studied. Understanding the cellular and molecular regulation mechanisms of pro-inflammatory cytokines has a potential therapeutic significance by providing strategies for inducing or inhibiting desired cytokine profiles in response to dengue virus as it has recently been suggested (Eigle et al. 1997).
To Drs Hermman MG Schatzmayr and Marilda M Siqueira for their continuous encouragement.
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