INCREASED LEVEL OF SERUM NITRIC OXIDE IN PATIENTS WITH ...

Am. J. Trop. Med. Hyg., 66(6), 2002, pp. 762–764 Copyright © 2002 by The American Society of Tropical Medicine and Hygiene

SHORT REPORT: INCREASED LEVEL OF SERUM NITRIC OXIDE IN PATIENTS WITH DENGUE NEREIDA VALERO, LUZ M. ESPINA, GERMAN AÑEZ , ENRIQUE TORRES, AND JESÚS A. MOSQUERA Sección de Virología and Sección de Inmunología y Biología Celular, Instituto de Investigaciones Clínicas “Dr. Américo Negrett,” Facultad de Medicina, Universidad del Zulia, Maracaibo, Venezuela

Abstract. Nitric oxide (NO) has been involved in several infectious diseases. Virus dengue is capable of inducing increased levels of NO when cocultured with human Kupffer and spleen cells. However, no reports describe the levels of NO in patients with dengue infection. Increased levels of NO were found in patients with the classic form of the disease; however, in the hemorrhagic form of the disease, similar levels to those of healthy controls were found. In vitro studies showed no increased levels of NO when human platelets were incubated with the virus. Increased NO in classical dengue could be important in the evolution from the nonhemorrhagic to the hemorrhagic forms of dengue. cubated with 50 ␮L of Griess reagent (0.5% sulfanilamide, 0.05% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4) and incubated for 10 minutes at room temperature. The lower limit detection of the assay was 1.5 ␮M. The optical densities of the samples were then obtained on a microplate reader (Benchmaru; Bio-Rad, Hercules, CA) at 540 nm. A standard curve that used NaNO2 was used to calculated sample concentrations (expressed as ␮M/L). We determined the effect of dengue virus on NO production by assessing normal human platelets. Platelet assays were performed in 1 mL of platelet-virus suspension in human plasma at 250,000 platelets/mL and at a multiplicity of infection of 0.5. The multiplicity of infection used to infect platelets was chosen from previous research in which the authors used a range of multiplicity of infection from 0.1 to 2 to induce several experimental conditions.8–10 Platelet-poor plasma from a healthy donor was used to prepare an adequate platelet concentration. Platelet-virus suspensions were incubated for 1, 4, and 6 hours at 37ºC. Dengue virus (New Guinea C strain) was used in active and inactive (80°C for 1 hour) forms. After centrifugation, supernatants were assayed for nitrite and nitrate content as previously described. As a positive control, we used platelets from people without virus but treated with 0.1 U/mL of bovine thrombin. In this way, we found values of 52.23 ± 13.4 ␮M/L of nitrate and nitrite in culture supernatants. Data are expressed as mean ± standard deviation. Differences between groups were analyzed by analysis of variance. The statistical significance set at P < 0.05. High concentration of nitrite or nitrate was observed in the patients with DF (P < 0.01) when compared with patients with DHF and with controls (Figure 1). No differences were observed between values from patients with DHF and controls. Values according age or sex in DF, DHF, or control groups were statistically not significant, suggesting that the differences observed between groups in our study were unrelated to age or sex. There were no statistical differences in serum nitrite or nitrate levels related to viral serotype infection. Patients with DF who were infected with serotype 4 had levels of nitrite or nitrate similar to the patients infected with serotype 1 (55.6 ± 2.25 ␮M/L and 57.4 ± 3.02 ␮M/L, respectively). For patients with DHF, a similar findings were observed (serotype 2, 13.87 ± 2.05 ␮M/L; serotype 4, 14.33 ± 1.25 ␮M/L). The increased production of NO in patients with DF is an expected finding because dengue virus is capable of inducing NO in cultures of spleen and Kupffer cells.2,3 Because plate-

INTRODUCTION Dengue is an acute mosquito-transmitted viral disease characterized by a mild febrile illness known as dengue fever (DF). Some infections result in dengue hemorrhagic fever (DHF), a syndrome that in its most severe form can threaten the patient’s life, primarily through increased vascular permeability and shock.1 The pathogenesis of DF and DHF is not fully understood. Previous reports have shown increased production of nitric oxide (NO) in cultures of Kupffer and spleen cells and in mice infected with dengue virus.2–4 These observations raise the possibility that increased NO production could be present in the patient during the course of dengue infection. In order to gain further information about this, we determined the serum concentration of NO in patients with DF and DHF; in addition, we also determined the NO production in human platelets cultured with dengue virus. This study was carried out in 105 patients admitted to the Regional Health System of Zulia State, Venezuela, and to the section of virology at Instituto de Investigaciones Clinicas “Dr. Americo Negrette,” Faculty of Medicine, Zulia, Venezuela. The patients were clinically and serologically classified as having DF (n ⳱ 66; platelet count, 168,200 ± 17,950/mm3) and DHF without shock (n ⳱ 39; platelet count, 63,570 ± 28,280/ mm3). Patients having DHF were classified as grade 1 and 2 on the basis of the criteria of the World Health Organization.5 In addition to clinical evidences the diagnosis was confirmed by the determination of specific serum immunoglobulin (Ig) M and IgG. Age-matched controls (n ⳱ 53) represent normal, healthy individuals. Studies were performed on dengue sera taken 1 to 8 days after onset of the disease. Sera from patients and controls were rapidly separated and stored at −70°C until use. The presence of specific IgM and IgG against the different dengue virus serotypes (types 1, 2, 3, and 4) were determined by immunoenzymatic assay (dengue IgG and IgM capture enzyme-linked immunosorbent assay kit; Pan Bio, Brishane, Australia). The method for dengue virus identification in infected cell cultures by use of specific monoclonal antibodies was indirect immunofluorescence.6 Virus isolated from serum samples were serotypes 1, 2, and 4. One hundred percent of the patients with DHF had secondary infections. Patients with DF had 92% primary and 8% secondary infections. Platelet values were determined with an automatic cell counter (Coulter Differential, Miami, FL). Total production of NO was determined by assaying for nitrite and nitrate.7 Briefly, 50 ␮L of serum from patients and controls were in-

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DENGUE AND NITRIC OXIDE

FIGURE 1. Increased level of seric nitrite and nitrate in patients with dengue fever (DF). Similar levels were found in patients with dengue hemorrhagic fever (DHF) and healthy controls. *P < 0.01 when DF is compared with DHF and control. Data represent mean ± standard deviation.

lets can generate NO through stimulation of NO synthase,11,12 we performed experiments to determine the production of NO after coculture of human platelets with active and inactive forms of dengue virus type 2. Virus-platelet interaction did not contribute to increased levels of NO in the culture supernatants (Table 1). These data suggest that platelets are not a source of NO during the course of DF. Levels of NO in the severe form of illness (DHF) were not found to be increased; they were similar to the normal serum levels (Figure 1). This suggests that NO could play a role in the course of the disease. Previous research has shown that NO induces vasorelaxation with reduced systemic vascular resistance and therefore blood pressure,13 and excessive NO production could induce shocklike syndrome.14 The complex events that address the vascular leakage and hemorrhagic manifestations in DHF6 could be modified by NO. In this regard, several reports indicate that NO can inhibit viral replication,15,16 and it has been demonstrated to play a role in the transmission of dengue virus–specific suppressor signal.17 In addition, NO has been involved in the innate immunity against dengue-infected cells.18 Previous reports have proposed that dengue virus infection of monocyte or macrophages is increased by antibodydependent enhancement, and the higher number of dengue virus-infected monocytes or macrophages results in increased T-cell activation, which results in the release of increased levels of cytokines, which in turn may lead to hemorrhage.6,19,20 The antiviral effect of NO could reduce those events and protect cells against deleterious effects of cyto-

TABLE 1 Nitrite and nitrate production in dengue serotype 2 (DEN-2) virustreated human platelets* Time after infection (h) Virus

1

4

6

Control DEN-2 Inactivated DEN-2

9.50 ± 1.34 8.61 ± 1.17 8.55 ± 1.06

10.08 ± 0.82 11.43 ± 1.22 8.87 ± 1.05

13.04 ± 2.82 10.84 ± 0.85 11.67 ± 1.71

* No significant differences were observed between groups (n ⳱ 10). Positive controls represent platelet cultures treated with 0.1 U/mL of bovine thrombin; the value of nitrate or nitrite observed was 52.23 ± 13.4. Values are expressed as ␮M/L.

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kines. Homeostatic changes in DHF involve vascular changes, thrombocytopenia, and disseminated intravascular coagulation.6,20,21 In addition, platelets have been shown to have increased binding to dengue-infected endothelial cells in vitro, suggesting a mechanism for thrombocytopenia.22 In this regard, NO can inhibit platelet aggregation23 and adhesion.24 These effects could contribute to the prevention of endothelial damage during the course of DF. Endothelial cells are one of the targets of dengue viruses. Dengue serotype 2 virus has been shown to have the highest virus replication in human cultured endothelial cells; it is capable of inducing apoptosis, complement activation, and chemokine production.8,25,26 Because it has been shown that apoptotic death after viral infection can trigger inflammatory processes,27 the antiviral effect of NO could interfere with the dengue virus apoptotic effect and its further deleterious events.25 The pathogenesis of DHF is not well known, but the role of host factors has been suggested. A strong correlation between high concentration of tumor necrosis factor alpha (TNF-␣) in blood and the severity of DHF has been reported.28–31 The presence of apoptosis has been suggested to be related to the pathogenesis of DHF,25,32 and TNF-␣ could be involved in the pathogenesis of DHF by inducing apoptotic processes.33 NO can protect cells from apoptosis induced by TNF-␣,34 and in addition, NO has an inhibitory effect on TNF-␣ release from human peripheral blood monocytes.35 Both protective mechanisms could play a role in DF patients with high levels of NO. In Venezuela, in previous epidemic reports of DHF, the disease has been associated with dengue serotype 2 in accordance with, in this study 80% of cases of DHF were associated with serotype 2 (serotype 4, 20%).36 Interestingly, a high percentage of the patients with DF (53) were also positive for serotype 2 (serotype 4, 45%; serotype 1, 2%); however, they had higher levels of NO, suggesting that virus serotype is not involved in the increased production of NO. The reason for the basal levels of NO in patients with DHF is not clear to us, but damage of endothelial cells and failure of endothelial NO synthase could be involved. These observations imply that increased levels of NO in DF could alter the evolution of the disease to its hemorrhagic form. Acknowledgment: We thank Dr. Duane Gubler (Fort Collin Centers for Disease Control and Prevention, Fort Collins, Colorado) for the monoclonal antibodies against dengue virus serotypes. Financial support: This study was supported by the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICIT), grant S197001133. Authors’ addresses: Luz M. Espina, Nereida J. Valero, and German Añez, Seccion de Virologia, Instituto de Investigaciones Clinicas “Dr. Americo Negrette,” Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela. Enrique Torres, Seccion de Hematologia, Instituto de Investigaciones Clinicas “Dr. Americo Negrette,” Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela. Jesús A. Mosquera, Seccion de Inmunologia y Biología Celular, Instituto de Investigaciones Clinicas “Dr. Americo Negrette,” Facultad de Medicina, Universidad del Zulia, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela. Reprint requests: Jesús A. Mosquera, Apartado Postal 1151, Maracaibo 4001-A, Zulia, Venezuela, Telephone, 58-61-597247, Fax: 5861-597247, E-mail: [email protected].

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