Blood Cells, Molecules, and Diseases 45 (2010) 334335

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Blood Cells, Molecules, and Diseases

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d

Letter to the Editor

Tissue factor expression on monocytes from patients with severe dengue fever Peripheral blood mononuclear cells (PBMCs) from 11 healthy individuals and 33 dengue infected patients were obtained from 20 mL of heparinised venous blood. Blood samples were processed according to earlier descriptions [2,8]. Briey, PBMCs were stored at liquid nitrogen after density gradient isolation from blood. Thawed PBMCs were labelled for ow cytometry analysis. CD14+ monocytes from dengue infected patients were labelled ex vivo according to previously described methods [2,8] with slight modications. Labelling was performed to conrm that 95% of the monocyte gated cells would be CD14+. PBMCs were labelled with monoclonal anti-human TF (Calbiochem, San Diego, CA, USA) for 30 min at 4 C in dilutions recommended by the manufacturer and further incubated with anti-mouse IgG labelled Alexa 488 (Molecular Probes, Invitrogen, Eugene, Oregon, USA) for 30 min. Finally, cells were ressuspended in 1% paraformaldehyde and kept at 4 C up to 24 h until ow cytometry. Cells were acquired (10,000 for gated monocytes) on a FACS Calibur ow cytometer (BD Biosciences) and analysed by FlowJo Software version 4.3 (TreeStar Inc., San Diego, CA, USA). Isotype-matched antibodies were used as a negative control for all labelling procedures. Dengue patients were studied for prior incidence of infection, detected by serologic immune response. All patients with mild disease were characterised as having no IgG antibodies for the virus, classied therefore as primary infection. Patients with a prior dengue infection were more likely to be experiencing severe disease (7 out 11), although no statistical signicance was found for differences observed between immune status (primary and secondary infection) or infecting virus serotype. Patients with severe disease, compared to patients with mild disease, had a signicant reduction in platelet counts during acute phase of infection (severe disease, 34 24 10 9 /L vs. mild disease, 194 92 109/L, respectively). Here, we show that TF expression on monocytes is markedly increased in patients with severe dengue fever in the acute phase of the disease when compared with patients with mild disease and healthy individuals (Fig. 1). We observed a signicant negative correlation between the platelet counts and the TF-expressing monocytes (Spearman correlation r = 0.70 with P = 0.0002, calculated by Prism 4). Monocytes may play a central role in the course of dengue fever. We reported increased frequencies of proinammatory monocytes (CD16+) and production of inammatory factors in the acute phase of disease [8]. During mild dengue fever these cells may express toll-like receptors (TLRs) that are associated with a good prognosis and might trigger antiviral and protective molecules. Monocytes are recognized as one of the main sources of blood-borne TF [5]. The coagulopathy observed during dengue fever may be caused by multiple factors [6,9]. This study shows for the rst time that monocytes from patients with severe dengue fever express high TF levels. Thus, monocytes are activated during the disease and, besides controlling infection, TF is expressed and released from infected cells contributing to the coagulation disorder and thrombocytopenia.

Dengue fever is a public health problem worldwide. The majority of dengue cases have a mild self-limited clinical course, whereas a small proportion progress to severe disease that is characterised by homeostatic abnormalities, including plasma leakage and bleeding [1]. The mechanisms leading to severe illness are not well understood but they are thought to involve an intricate interplay between virus, vascular cells, and inappropriate host immune responses. Mononuclear phagocytes are considered main targets for dengue virus replication. The infection of monocytes may induce inammatory mediators and is the source of viral dissemination in the initial phase of the disease [2]. Tissue factor (TF) is a 47-kD protein that initiates the clotting cascade and is increasingly recognized at the interface of blood coagulation and inammation [3,4]. Activation of coagulation by TF is frequently observed in the sepsis syndrome and has been proposed to play a role in certain viral hemorrhagic fevers [4]. TF binds to factor VII (FVII)/FVIIa and the binary TF/FVIIa complex initiates the coagulation cascade upon activation of factors IX and X, which leads to thrombin generation and platelet activation. Under physiological conditions TF is absent in blood. However, upon cytokine stimulation some cells in particular monocytes and endothelial cells may express this molecule [5]. Coagulation abnormalities occur during viral hemorrhagic fevers such Ebola and dengue [6,7]. Moreover, dendritic cells and macrophages are susceptible to Ebola virus, leading to TF upregulation, brin generation, and microthrombosis [7]. Dengue hemorrhagic fever, a severe form of dengue fever, is associated with increased TF plasma levels but the cellular origin remains speculative [6]. In the present investigation dengue infected patients (n = 33) and healthy individuals (controls, n = 11) were enrolled. Patients were assisted at two Brazilian Health Centres (Hospital Universitrio Professora Esterian Corsini, UFMS, MS, and Hospital Universitrio Pedro Ernesto, UERJ, RJ). All patients had a clinical diagnosis of dengue infection according to the criteria of the World Health Organization /Brazilian Health Ministry. Nineteen patients were considered to have mild dengue fever because no warning signs (WS) were observed, only classical symptoms of dengue fever. Fourteen individuals were considered to have severe disease consisting of those who presented severe clinical manifestations such as haemoconcentration, thrombocytopenia (counts b 50 109/L), plasma leakage or internal haemorrhages. Criteria were based in earlier studies [8]. Dengue virus infection was conrmed either by anti-dengue enzyme-linked immunoabsorbent assay (ELISA)-IgM, serotype specic reverse transcriptionpolymerase chain reaction (RT-PCR) or by virus isolation. The study was approved by Ethical Committees at FIOCRUZ (IPEC, FIOCRUZ CAAE 3723.0.000.009-08). Blood was collected after written informed consent.
1079-9796/$ see front matter. Published by Elsevier Inc. doi:10.1016/j.bcmd.2010.08.004

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Fig. 1. Monocytes from severe DF patients express TF during acute phase of disease. Peripheral blood monocyte cells from healthy subjects (n = 11) or samples from dengue mild DF patients (n = 19) and severe DF patients (n = 14) were labelled with specic mAbs and analysed by single color ow cytometry for TF expression. Statistical signicance was assessed by non-parametric KruskalWallis test followed by Dunn's Multiple Comparison Test. *** P b 0.001, calculated by Prism 4 (http://www. graphpad.com).

Elzinandes Leal de Azeredo Claire Fernandes Kubelka Lidiane Martins Alburquerque Luciana Santos Barbosa Laboratrio de Imunologia Viral, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, CEP 21045-900, Brazil E-mail address: elzinandes@ioc.ocruz.br (E.L. de Azeredo). Corresponding author. Fax: + 55 21 22604866. Paulo Vieira Damasco Carlos Andr Lins vila Hospital Universitrio Gaffree e Guinle, UNIRIO, Rio de Janeiro, Brazil Universidade Estadual do Rio de Janeiro, UERJ, Rio de Janeiro, Brazil Ana Rita C. Motta-Castro Rivaldo Venncio da Cunha Setor Hemoncleo, CCBS, Universidade Federal do Mato Grosso do Sul, UFMS, Mato Grosso do Sul, Brazil Departamento de Clnica Mdica, FM, Universidade Federal do Mato Grosso do Sul, UFMS, Mato Grosso do Sul, Brazil Robson Q. Monteiro Instituto de Bioqumica Mdica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 20 July 2010

Acknowledgments This work was supported by Instituto Oswaldo Cruz (IOC), Universidade Federal do Rio de Janeiro (UFRJ), Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq), and Fundao de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ).

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