Short communicationEpstein-Barr virus infection of human brain microvessel endothelial cells: A novel role in multiple sclerosis
Introduction
Despite decades of focused biomedical research, the etiology of multiple sclerosis (MS) is currently not well understood. Epidemiological data and geographic patterns imply that MS is caused by an environmental insult in genetically predisposed individuals (Sospedra and Martin, 2005). Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of MS. The risk of MS is 20 times greater among people who have contracted infectious mononucleosis, a disease caused by EBV, as compared with seronegative individuals (Ascherio and Munger, 2007). A steep increase in the titer of antibodies specific for EBV has been detected in the serum of MS patients: it begins to elevate 10–20 years prior to the onset of the symptoms and correlates with the risk of developing the disease (Levin et al., 2005). CD4+ T cells that cross-react with both EBV and myelin basic protein and co-produce IFN-γ and IL-2 have been isolated from MS patients (Lunemann et al., 2008). Interestingly, a recent study detected a decreased T cell reactivity against EBV infected B cells in MS patients indicating a dysregulated EBV immune response (Pender et al., 2009). Despite this evidence, the mechanism by which EBV would trigger MS remains elusive (Lunemann and Munz, 2009). Intriguingly, the infection of endothelial cells by other herpes viruses such as cytomegalovirus or Kaposi's sarcoma virus up-regulates the expression of cytokines, chemokines and adhesion molecules (Valbuena and Walker, 2006). Taking a fresh approach, we reasoned that EBV infection of atypical target cells such as endothelial cells may be an essential component to pathogenesis in MS.
In transgenic TCR mouse models, MS is triggered only after the blood-brain barrier (BBB) is compromised (Brabb et al., 1997). The presence of autoreactive myelin-specific lymphocytes is not sufficient to cause MS since such cells have been isolated from healthy individuals (Ota et al., 1990). In MS patients, lesion and plaque formations are associated with a disruption of the BBB. Thus, an increase in BBB permeability, along with autoreactive T cells activation, is a requirement for the development of MS. However, it is unclear how changes in the BBB are initiated prior to the initial immune cell entry into the brain (Ransohoff, 2009).
We hypothesized that EBV infection of a subset of brain endothelial cells would increase the potential for an inflammatory breach of the BBB, particularly following reactivation of latent virus. Prior work has established that EBV can infect macrovascular endothelial cells both in human tissues (Ban et al., 1999, Guarner and Unger, 1990) and in culture with human umbilical vein endothelial cells (HUVECs) (Jones et al., 1995, Xiong et al., 2004). However, the BBB is composed of microvascular endothelial cells that differ significantly from macrovascular endothelial cells, such as HUVECs, in a number of characteristics, most importantly in terms of susceptibility to viral infection by Herpesviridae (Jarvis and Nelson, 2007). To investigate our premise, we asked whether EBV could infect primary human brain microvascular endothelial cells (HBMECs) leading to increased endothelial cell activation that would be relevant for MS pathogenesis.
Section snippets
Cell-lines
Primary cultures of HBMECs were established from normal brains at autopsy as previously described (Dorovini-Zis et al., 1991). The endothelial origin and purity of these cells were confirmed by the strong positive staining for Factor VIII-related antigen and binding of Ulex europeaus agglutinin. Primary HBMECs cultures retained important morphological and functional characteristics of their in vivo counterparts, mainly, interendothelial tight junctions that are impermeable to proteins, high
Results and discussion
HBMECs isolated from three different donors were successfully infected with EBV (Fig. 1A) under standard laboratory culture conditions. The viral genome was detected by standard PCR in infected HBMECs and was absent from mock-infected HBMECs, indicating that the donors' endothelial cells were EBV negative (Fig. 1A). EBV infection did not diminish the viability of HBMECs and this was confirmed using esterase activity assays (data not shown). Further, EBV gene expression was detected by RT-PCR 9
Acknowledgements
This research was supported by the Multiple Sclerosis Society of Canada. The authors want to thank Rukmini Prameya for technical assistance with the culture of brain endothelial cells and PBMCs isolation.
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