In contrast to the case for seasonal influenza virus, human infection with HPAI H5N1 viruses causes severe disease of the lower respiratory tract, and many patients progress rapidly to ARDS and multiorgan failure (16
). Although endothelial cell injury is a general characteristic feature of ARDS (51
), the role of pulmonary endothelial cells in H5N1 pathogenesis is still largely unknown. Here, we provide data obtained using a pulmonary endothelial cell line and primary cells isolated from human lung tissue and compare host cell permissiveness to infection with influenza viruses of multiple subtypes. The human pulmonary endothelial cell model was also utilized to evaluate the cell receptors for influenza virus and innate immune responses induced by HPAI H5N1 virus compared with other influenza virus subtypes. We found that unlike human bronchial epithelial cells, which are permissive to productive replication of both avian and human influenza viruses and express avian-like α-2,3-linked and human-like α-2,6-linked SA receptors equally well (7
), pulmonary endothelial cells possess α-2,3-linked SA configuration as the dominant receptor type and support efficient replication only of HPAI H5N1 virus. The polybasic HA cleavage site was found to be necessary but not sufficient for the high replication efficiency of HPAI H5N1 virus in pulmonary endothelial cells. The H5N1 virus replication correlated with high-level expression of proinflammatory cytokines/chemokines and adhesion molecules and also resulted in a marked decrease in cell viability and proliferation, which could be attributable to virus-mediated cell death and/or a cytokine-mediated process.
The clinical course of H5N1 virus infection in humans often results in the progression of severe lower respiratory tract disease, which includes dyspnea, chest pain, and pulmonary infiltrates (1
). Infiltration of neutrophils, alveolar edema, endothelial hypertrophy, and necrosis among H5N1-infected patients are thought to contribute to the severe lung pathology of fatal human infection. Although in situ
hybridization failed to show positive viral RNA or influenza NP antigen staining in lung endothelial cells from postmortem examination of patients who succumbed to H5N1 infection (19
), the detection of IFN-β-positive endothelial cells in lungs of H5N1-infected patients suggests that these cells are responding to viral infection (17
). However, in situ
evidence for H5N1 virus infection of pulmonary endothelial cells in humans is limited due to the paucity of postmortem tissues for study. In the macaque model, endothelial cells appeared to be productively infected and displayed necrosis in H5N1-challenged animals (4
). In standard cultures, we observed that both human and rat pulmonary microvascular endothelial cells could be productively infected by VN/1203 (H5N1) virus, which was highly lethal in ferrets and mice (29
). Utilizing a polarized endothelial cell model, in which cells were seeded on transwells creating two distinct surfaces, i.e., apical and basolateral domains, we found preferential viral entry and release from the apical surface. HPAI H5N1 virus particles bud from the apical (luminal) side of endothelial cells, in contact with blood, so that released virus could enter the general circulation and cause viremia. Unlike for seasonal influenza viruses, which are confined mainly to the upper respiratory tracts of mammals, viral RNA has been detected in the blood of H5N1-infected patients with extrapulmonary complications, including multiorgan failure, which are common in fatal cases (reviewed in reference 46
). It is reasonable to speculate that efficient replication of H5N1 virus in pulmonary endothelial cells may promote systemic spread of virus and contribute to the pathogenicity of HPAI H5N1 viruses in the mammalian host. Since transport of H5N1 virus from the basolateral to the apical chamber was minimal in the polarized endothelial cell model, it is not entirely clear how initial apical infection occurs in these cells.
In this study, we have addressed the possible contribution of the HA glycoprotein to the replication efficiency of H5N1 virus in pulmonary endothelial cells. The two main functions of the HA are to facilitate (i) virus binding to target host cells, via sialic acid-containing receptors, and (ii) virus entry into the cell, which is dependent on HA cleavage. Because the availability of suitable receptors on the host cell surface can determine efficiency of influenza virus infection and replication (41
), the abundance of surface-associated SA on pulmonary endothelial cells was analyzed through careful flow cytometric analysis coupled with lectin staining in a dose-dependent manner. It has been reported that α-2,3-SA and α-2,6-SA receptors were homogenously expressed on microvascular endothelial cells (52
). However, we found that the α-2,3-SA configuration was much more abundant than α-2,6-SA receptors, which corresponded to a lower rate of infectivity for human influenza viruses possessing the α-2,6-SA receptor binding preference. Moreover, avian-origin viruses with preferential α-2,3-SA binding attached to and infected pulmonary endothelial cells at a higher frequency than human influenza viruses. Taken together, the data suggest that the receptor specificity most likely contributes to the efficient H5N1 virus replication in these cells; however, additional data are needed to definitively assess the role of this molecular determinant. The generation of mutant influenza viruses that carry substitutions in the H5 HA receptor binding site conferring a change in binding preference from the avian- to the human-type receptor is required.
The cleavage properties of HA0
and the distribution of functional proteases in the host contribute to tissue tropism (14
). Unlike airway epithelial cells (54
), pulmonary endothelial cells do not appear to produce sufficient endogenous proteases capable of cleaving HA at a monobasic cleavage site. We found that these cells support multiple-cycle growth only of H5N1 viruses containing a polybasic cleavage site. Removal of the polybasic cleavage site in VN/1203 virus did not affect the initial viral infectivity in pulmonary endothelial cells; however, it greatly attenuated viral growth kinetics relative to that of wild-type virus. The results lend support to the concept that in vivo
, the polybasic HA cleavage site is a molecular determinant not only for extrapulmonary virus replication but also for enhancing H5N1 virus replication in pulmonary endothelial cells. H5N1 infection of endothelial cells may contribute to the high viral load in lung tissue and potential virus spread beyond the respiratory tract.
Although HA appears to play an important role in efficient H5N1 virus replication through its receptor binding specificity and polybasic cleavage site, additional virulence determinants of the virus most certainly exist. We observed that a reassortant virus containing six internal genes from A/PR/8/34 (H1N1) and the HA and NA genes from VN/1203 virus exhibited attenuated replication in pulmonary endothelial cells compared with wild-type VN/1203 virus despite comparable efficiencies of initial infectivity (based on NP staining) (data not shown). Moreover, H7 subtype viruses and the less virulent SP/83 and Ck/Korea H5N1 viruses, which possess an “avian-like” α-2,3-SA binding preference and a polybasic cleavage site, could not productively replicate as efficiently as HPAI H5N1 viruses in human pulmonary endothelial cells, further suggesting that additional molecular factors are associated with HPAI H5N1 virus virulence.
ARDS is characterized by diffuse alveolar damage usually following an intense inflammatory response to infection (51
). The suggestion that the fatal outcome in patients with H5N1 virus infection may be attributed to elevated levels of proinflammatory cytokines (4
) prompted us to characterize the mediators of inflammation produced by human pulmonary endothelial cells. We found that H5N1 virus infection resulted in elevated production of multiple cytokines, including TNF, IP-10, IL-6, IL-8, MCP-1, IFN-γ, VEGF, and RANTES. TNF and IL-8 are of particular interest because of their association with ARDS and can instigate a cascade of physiological changes, including recruitment of neutrophils leading to alveolar capillary damage (51
). Moreover, it has been demonstrated that H5N1-infected, but not H1N1-infected, TNFR-1-deficient knockout mice exhibit a substantial reduction in lung inflammation and delay in mortality compared to wild-type mice, suggesting that TNF contributes to H5N1-induced inflammation of lung tissue (38
). Blocking of TNF has been shown to decrease vascular permeability through the destabilization of the endothelial cell cytoskeleton (47
) and to affect subsequent immune cell transmigration across the endothelium (26
). When exposed to TNF, a normally “quiescent” endothelium becomes activated and expresses additional proinflammatory factors, including chemokines and adhesion molecules (51
). In our analysis we also observed that human pulmonary endothelial cell cultures generated high levels of adhesion molecules, including selectin P ligand, selectins L and E, ICAM1, and VCAM1, in response to H5N1 but not H1N1 virus infection. Production of chemokines and adhesion molecules by endothelial cells could contribute to the tissue damage by causing vascular injury and destruction of the parenchymal cells through the accumulation of inflammatory cells (3
). The resulting loss of functional alveolar surface area could result in inadequate gas exchange, lower respiration, and ultimately death.
The pulmonary endothelium is strategically located within the lung, and its functional and structural integrity are essential for adequate pulmonary function. The majority of patients with confirmed H5N1 virus infection have an aggressive clinical course and often present with respiratory failure frequently complicated with ARDS (44
). ARDS induced by H5N1 viral infection is most likely to be linked to patient death, and pulmonary endothelial injury is expected to contribute to the abnormalities seen in H5N1-induced ARDS. Our results demonstrate a unique virulence trait of HPAI H5N1 following virus infection of pulmonary endothelial cells which leads to a high virus load, an overwhelming immune reaction, and a marked decrease in cell viability. Future investigation of the interaction between H5N1-induced inflammation and the pulmonary endothelium, especially in in vivo
models, is warranted.