Increased permeability of human endothelial cell line EA.hy926 induced by hantavirus-specific cytotoxic T lymphocytes
Introduction
Hantaviruses, belonging to the family Bunyaviridae, are distributed worldwide. Two forms of zoonotic human diseases are caused by hantavirus species (Enria et al., 2001, Linderholm and Elgh, 2001, Schmaljohn and Hjelle, 1997). Hemorrhagic fever with renal syndrome (HFRS) and its mild form nephropathia epidemica (NE) are caused by Old World (Europe and Asia) hantaviruses, such as Hantaan, Seoul, Dobrava and Puumala viruses. Hantavirus pulmonary syndrome (HPS) is caused by New World (North and South America) hantaviruses, such as Sin Nombre and Andes viruses (Khaiboullina and St Jeor, 2002, Zeier et al., 2005). The hantavirus genome consists of three RNA segments, large, medium and small segments, encoding RNA-dependent RNA polymerase, envelope glycoproteins (G1 and G2), and nucleocapsid (N) protein, respectively (Jonsson and Schmaljohn, 2001). Hantaviruses persistently infect their natural rodent reservoirs without apparent diseases (Meyer and Schmaljohn, 2000). Humans are infected with hantaviruses by direct contact with infected rodents or through the inhalation of excreted viral aerosols (Linderholm and Elgh, 2001).
Human hantavirus diseases are characterized by an increased permeability in microvascular beds of the kidneys in HFRS and the lungs in HPS, and endothelial cells are considered to be the primary targets of hantavirus infection (Hautala et al., 2002, Khaiboullina and St Jeor, 2002, Mustonen et al., 1994, Temonen et al., 1996, Zaki et al., 1995). In vitro hantavirus infection alone, however, did not induce visible cytopathic effects in cultured human endothelial cells (Geimonen et al., 2002, Niikura et al., 2004, Pensiero et al., 1992) nor did it increase capillary permeability of an infected endothelial cell monolayer (Khaiboullina et al., 2000, Niikura et al., 2004, Sundstrom et al., 2001). Increased levels of cytokines including tumor necrosis factor (TNF) -α, interleukin (IL) -2, IL-6 and interferon (IFN) -γ have been detected in sera of HFRS and HPS patients (Khaiboullina and St Jeor, 2002, Vapalahti et al., 2001). An expanded leukocyte population including monocytes, T and B cells and an increase of CD8+/CD4+ ratio have been observed in HFRS patients (Chen and Yang, 1990, Huang et al., 1994, Lewis et al., 1991, Markotic et al., 1999). We found significantly higher frequencies of Sin Nombre virus (SNV)-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8+ T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8+ T cells). These results suggest that virus-specific CD8+ T cells contribute to HPS disease outcome (Kilpatrick et al., 2004). Increased numbers of CD8+ T cells are found in the kidneys of HFRS (Temonen et al., 1996) and in the lungs of HPS patients (Zaki et al., 1995). There is an abundance of immune cells expressing a variety of cytokines in the lungs of HPS cases (Mori et al., 1999, Zaki et al., 1995) and in the kidneys of NE cases (Temonen et al., 1996). In addition, preliminary evidence suggests that HLA-B*3501 is associated with severe HPS in SNV infection, implying involvement of CD8+ cytotoxic T lymphocytes (CTLs) (Kilpatrick et al., 2004) (Koster et al., 2001). A similar linkage between disease severity and MHC haplotype was observed between Puumala virus infection and the HLA-B8-DR3 extended haplotype (severe outcome) or the HLA-B27 (milder disease) (Makela et al., 2002, Mustonen et al., 1998). These reports suggest that host immune responses against hantavirus, especially virus-specific CTLs and inflammatory cytokines produced by virus-specific T cells may contribute to disease pathogenesis of HPS and HFRS (Terajima et al., 2004).
We have established panels of CTL lines from the PBMC of NE and HPS patients (Ennis et al., 1997, Kilpatrick et al., 2004, Terajima et al., 2002, Terajima et al., 2004, Van Epps et al., 1999, Van Epps et al., 2002). These cell lines were able to recognize and lyse autologous B lymphoblastoid cell lines pulsed with the epitope peptide or infected with recombinant vaccinia viruses expressing the hantaviral protein containing the epitope. In this report, we tested one of these CTL lines against human endothelial cell line, EA.hy926, infected with SNV in vitro. Primary human umbilical vein endothelial cells (HUVECs) or human lung microvascular endothelial cells (HMVEC-Ls) have been used to examine the effect of hantavirus infection on endothelial cell function (Gavrilovskaya et al., 2002, Khaiboullina et al., 2000, Niikura et al., 2004, Sundstrom et al., 2001). Although ideally the interaction between hantavirus-specific CTL and hantavirus-infected endothelial cells should also be analyzed using HUVECs or HMVEC-Ls, it is difficult to obtain endothelial cells that express the MHC class I molecules by which our CTL lines were restricted. Therefore, we used the immortalized human endothelial cell line EA.hy926 (Edgell et al., 1983), which we found to express the HLA-A24 allele (in this report), and one of our hantavirus-specific CTL line, 1A-E2, which was established from convalescent PBMC from the patient infected with Puumala virus and was restricted by HLA-A24 (Terajima et al., 2002). 1A-E2 recognized the epitope, HWMDATFNL, encoded by Puumala virus G2 protein, and was cross-reactive to the corresponding peptide, HWMDGTFNI, in SNV G2 protein (Terajima et al., 2002). EA.hy926 cells are the most similar to HUVEC among the available immortalized human endothelial cell lines and have been used to study the endothelial cell/leukocyte interactions (Lidington et al., 1999).
In this study, we first tested the infectivity of SNV to EA.hy926 cells. Next, we showed that the hantavirus-specific CD8+ T cell line 1A-E2 could recognize and lyse EA.hy926 cells infected with SNV or presenting SNV antigen. Finally, we demonstrated that 1A-E2 enhanced the permeability of EA.hy926 cells infected with SNV. These results suggest that virus-specific CTLs contribute to the capillary leakage.
Section snippets
Virus and cell lines
SNV stock virus (strain CC107, kindly provided by Connie S. Schmaljohn) (Schmaljohn et al., 1995) was propagated in Vero E6 cells and aliquots were stored in −80 °C. All experiments using cultured live SNV were performed in biosafety level 3 laboratory of University of Massachusetts Medical School according to standard BSL3 guidelines. The EA.hy926 cell line, which had been derived by fusing HUVECs with the permanent human cell line A549, were kindly provided by Cora-Jean S. Edgell, University
SNV infection in EA.hy926 cells
It has been reported that HUVECs were permissive to hantavirus infection (Pensiero et al., 1992). We first tested whether immortalized human endothelial cell line, EA.hy926, would be infected with SNV. Confluent EA.hy926 cells and Vero E6 cells were inoculated with SNV at m.o.i. of 0.0025, and cell lysates and supernatants were harvested at 0, 1, 4, 8 and 12 days post-infection. To detect viral protein expression, Western blotting was performed using cell lysates (Fig. 1A). In both EA.hy926 and
Discussion
We demonstrated that hantavirus-specific CTLs increased the permeability of SNV-infected EA.hy926 cell monolayer after recognition of the antigen presented on cell surface. By using the recombinant replication-deficient adenovirus, which did not increase permeability of infected EA.hy926 cells, we show that CTLs alone were able to increase permeability after recognition of the antigen presented on infected cell.
We did not perform experiments analyzing the mechanisms of permeability change
Acknowledgments
We thank Cora-Jean S. Edgell of University of North Carolina for providing us EA.hy926 cell line, Connie S. Schmaljohn of the US Army Medical Research Institute of Infectious Disease for SNV strain CC107, Patrick C. Stockton and Thomas G. Ksiazek of the Centers for Disease Control and Prevention (CDC) for rabbit anti-SNV serum and Christina F. Spiropoulou for the pGEM-G2 plasmid. We thank Sunil K. Shaw of Vascular Research Division, Departments of Pathology, Brigham and Women's Hospital and
References (52)
- et al.
Human cytotoxic T lymphocyte responses to live attenuated 17D yellow fever vaccine: identification of HLA-B35-restricted CTL epitopes on nonstructural proteins NS1, NS2b, NS3, and the structural protein E
Virology
(2002) - et al.
Hantavirus pulmonary syndrome: CD8+ and CD4+ cytotoxic T lymphocytes to epitopes on Sin Nombre virus nucleocapsid protein isolated during acute illness
Virology
(1997) - et al.
DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection
Virology
(1999) - et al.
A lethal disease model for hantavirus pulmonary syndrome
Virology
(2001) - et al.
Apoptosis is induced by hantaviruses in cultured cells
Virology
(1999) - et al.
Changes in populations of immune effector cells during the course of haemorrhagic fever with renal syndrome
Trans. R. Soc. Trop. Med. Hyg.
(1991) - et al.
A comparison of primary endothelial cells and endothelial cell lines for studies of immune interactions
Transpl. Immunol.
(1999) - et al.
Persistent hantavirus infections: characteristics and mechanisms
Trends Microbiol.
(2000) - et al.
Hantavirus pulmonary syndrome in the United States: a pathological description of a disease caused by a new agent
Hum. Pathol.
(1995) - et al.
Isolation and initial characterization of a newfound hantavirus from California
Virology
(1995)
Cytokines, adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: an immunohistochemical study
Clin. Immunol. Immunopathol.
Generation of recombinant vaccinia viruses expressing Puumala virus proteins and use in isolating cytotoxic T cells specific for Puumala virus
Virus Res.
Immune responses to Puumala virus infection and the pathogenesis of nephropathia epidemica
Microbes Infect.
Abnormalities of T cell immunoregulation in hemorrhagic fever with renal syndrome
J. Infect. Dis.
Permanent cell line expressing human factor VIII-related antigen established by hybridisation
Proc. Natl. Acad. Sci. U.S.A.
Rapid cloning of HLA-A, B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification
Proc. Natl. Acad. Sci. U.S.A.
Clinical manifestations of New World hantaviruses
Curr. Top. Microbiol. Immunol.
Virology. Finally, a handle on the hantaviruses
Science
Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration
Arch. Virol.
Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses
Proc. Natl. Acad. Sci. U.S.A.
Hypophyseal hemorrhage and panhypopituitarism during Puumala Virus Infection: Magnetic resonance imaging and detection of viral antigen in the hypophysis
Clin. Infect. Dis.
Hemorrhagic fever with renal syndrome: relationship between pathogenesis and cellular immunity
J. Infect. Dis.
Replication of hantaviruses
Curr. Top. Microbiol. Immunol.
Class I, II and III HLA alleles associated with severe hantavirus cardiopulmonary syndrome in the southwest US
Am. J. Trop. Med.
Effects of tumor necrosis factor alpha on Sin Nombre virus infection in vitro
J. Virol.
Hantavirus immunology
Viral Immunol.
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Present address: Department of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.