Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.     

Dynamics of cellular immune responses in the acute phase of dengue virus infection

In this study, we examined the dynamics of cellular immune responses in the acute phase of dengue virus (DENV) infection in a marmoset model. Here, we found that DENV infection in marmosets greatly induced responses of CD4/CD8 central memory T and NKT cells. Interestingly, the strength of the immune response was greater in animals infected with a dengue fever strain than in those infected with a dengue hemorrhagic fever strain of DENV. In contrast, when animals were re-challenged with the same DENV strain used for primary infection, the neutralizing antibody induced appeared to play a critical role in sterilizing inhibition against viral replication, resulting in strong but delayed responses of CD4/CD8 central memory T and NKT cells. The results in this study may help to better understand the dynamics of cellular and humoral immune responses in the control of DENV infection.     

Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection

Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.     

Dengue virus infection: current concepts in immune mechanisms and lessons from murine models

Dengue viruses (DENV), a group of four serologically distinct but related flaviviruses, are responsible for one of the most important emerging viral diseases. This mosquito‐borne disease has a great impact in tropical and subtropical areas of the world in terms of illness, mortality and economic costs, mainly due to the lack of approved vaccine or antiviral drugs. Infections with one of the four serotypes of DENV (DENV‐1–4) result in symptoms ranging from an acute, self‐limiting febrile illness, dengue fever, to severe dengue haemorrhagic fever or dengue shock syndrome. We reviewed the existing mouse models of infection, including the DENV‐2‐adapted strain P23085. The role of CC chemokines, interleukin‐17 (IL‐17), IL‐22 and invariant natural killer T cells in mediating the exacerbation of disease in immune‐competent mice is highlighted. Investigations in both immune‐deficient and immune‐competent mouse models of DENV infection may help to identify key host–pathogen factors and devise novel therapies to restrain the systemic and local inflammatory responses associated with severe DENV infection.     

Dengue virus neutralization by human immune sera: Role of envelope protein domain III-reactive antibody

Dengue viruses (DENV) are the etiological agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). The DENV complex consists of four closely related viruses designated DENV serotypes 1 through 4. Although infection with one serotype induces cross reactive antibody to all 4 serotypes, the long-term protective antibody response is restricted to the serotype responsible for infection. Cross reactive antibodies appear to enhance infection during a second infection with a different serotype. The goal of the present study was to characterize the binding specificity and functional properties of human DENV immune sera. The study focused on domain III of the viral envelope protein (EDIII), as this region has a well characterized epitope that is recognized by strongly neutralizing serotype-specific mouse monoclonal antibodies (Mabs). Our results demonstrate that EDIII-reactive antibodies are present in primary and secondary DENV immune human sera. Human antibodies bound to a serotype specific epitope on EDIII after primary infection and a serotype cross reactive epitope on EDIII after secondary infection. However, EDIII binding antibodies constituted only a small fraction of the total antibody in immune sera binding to DENV. Studies with complete and EDIII antibody depleted human immune sera demonstrated that EDIII binding antibodies play a minor role in DENV neutralization. We propose that human antibodies directed to other epitopes on the virus are primarily responsible for DENV neutralization. Our results have implications for understanding protective immunity following natural DENV infection and for evaluating DENV vaccines.     

The pathology of dengue hemorrhagic fever

An estimated 2.5 billion people are at risk of dengue infection, and of the 100 million cases of dengue fever per year, up to 500,000 develop dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the life-threatening forms of the infection. The large majority of DHF/DSS occurs as the result of a secondary infection with a different serotype of the virus. While not completely understood, there is evidence that the target cells include dendritic reticulum cells, monocytes, lymphocytes, hepatocytes, and vascular endothelial cells. Viral replication appears to occur in dendritic cells, monocytes, and possibly circulating lymphoid cells, and damage to these and other target cells occurs through immune-mediated mechanisms related to cross-reacting antibodies and cytokines released by dendritic cells, monocytes, and vascular endothelium. There is evidence of a concomitant cellular activation as well as immune suppression during the infection. The activation of memory T cells results in cascades of inflammatory cytokines, including tumor necrosis factor-alpha, interleukins (IL-2, IL-6, and IL-8), and other chemical mediators that increase vascular endothelial permeability or trigger death of target cells through apoptosis. Pathological studies in humans are uncommon, and a suitable animal model of DHF/DSS does not exist. The current treatment of DHF/DSS is symptomatic, and prevention is through vector control. As such, there is a great impetus for the development of vaccines and novel therapeutic molecules to impede viral replication in infected cells or counteract the effects of specific inflammatory mediators on target cells. The role of genetics in relation to resistance to DHF/DSS also requires clarification.     

An improved endothelial barrier model to investigate dengue haemorrhagic fever

A cell culture model suitable for studies of dengue haemorrhagic fever was developed, based on culture of primary human umbilical vein endothelial cells (HUVECs) on a permeable membrane. By electron microscopy, cultured HUVECs at day 11 resembled morphologically microvascular endothelium. Endothelial barrier function was assessed by measuring transendothelial flux of albumin. Instead of using a labelled tracer molecule, an enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of native human albumin. The permeability characteristics of the HUVEC monolayer were found to be improved significantly (approximately 1 log reduction in permeability coefficient for albumin) by culturing HUVECs in human serum rather than fetal calf serum. Permeability coefficients for albumin in the range 1-4 x 10(-7) cm/s were achieved, which is an improvement on previous in vitro models of the endothelial barrier. Comparison of transendothelial flux of albumin and urea provided evidence of molecular sieving by the HUVEC monolayer. Moreover, tumour necrosis factor-alpha induced a dose-dependent, reversible increase in permeability of the HUVEC monolayer. This endothelial barrier model thus has many important characteristics that resembled human microvascular endothelium and is an improvement on the previous model proposed for studies of dengue haemorrhagic fever.     

Dengue hemorrhagic fever-associated immunomediators induced via maturation of dengue virus nonstructural 4B protein in monocytes modulate endothelial cell adhesion molecules and human microvascular endothelial cells permeability

We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.     

Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo

We used gene expression profiling of human primary cells infected in vitro with dengue virus (DENV) as a tool to identify secreted mediators induced in response to the infection. Affymetrix GeneChip analysis of human primary monocytes, B cells and dendritic cells infected with DENV in vitro showed strong induction of monocyte chemotactic protein 2 (MCP‐2/CCL8), interferon gamma‐induced protein 10 (IP‐10/CXCL10) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL/TNFSF10). The expression of these genes was confirmed in dendritic cells infected with DENV in vitro at mRNA and protein levels. A prospectively enrolled cohort of DENV‐infected Venezuelan patients was used to measure the levels of these proteins in serum during three different periods of the disease. Results showed significant increase of MCP‐2, IP‐10, and TRAIL levels in patients infected with DENV during the febrile period, when compared to healthy donors and patients with other febrile illnesses. MCP‐2 and IP‐10 levels were still elevated during the post‐febrile period while TRAIL levels dropped close to normal after defervescense. Patients with primary infections had higher TRAIL levels than patients with secondary infections during the febrile period of the disease. Increased levels of IP‐10, TRAIL and MCP‐2 in acute DENV infections suggest a role for these mediators in the immune response to the infection. MCP‐2 was identified in this work as a new unreported and important dengue‐related protein and IP‐10 was confirmed as a novel and strong pro‐inflammatory marker in acute disease. J. Med. Virol. 81:1403–1411, 2009. © 2009 Wiley‐Liss, Inc.     

Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model

The four serotypes of dengue virus (DENV1–4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-α/β and -γ receptors (AG129) were used to model secondary infection using both DENV1–DENV2 and DENV2–DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.     

Pathogenesis of vascular leak in dengue virus infection

Endothelial dysfunction leading to vascular leak is the hallmark of severe dengue. Vascular leak typically becomes clinically evident 3–6 days after the onset of illness, which is known as the critical phase. This critical phase follows the period of peak viraemia, and lasts for 24–48 hr and usually shows rapid and complete reversal, suggesting that it is likely to occur as a result of inflammatory mediators, rather than infection of the endothelium. Cytokines such as tumour necrosis factor‐α, which are known to be elevated in the critical phase of dengue, are likely to be contributing factors. Dengue NS1, a soluble viral protein, has also been shown to disrupt the endothelial glycocalyx and thus contribute to vascular leak, although there appears to be a discordance between the timing of NS1 antigenaemia and occurrence of vascular leak. In addition, many inflammatory lipid mediators are elevated in acute dengue viral infection such as platelet activating factor (PAF) and leukotrienes. Furthermore, many other inflammatory mediators such as vascular endothelial growth factor and angiopoietin‐2 have been shown to be elevated in patients with dengue haemorrhagic fever, exerting their action in part by inducing the activity of phospholipases, which have diverse inflammatory effects including generation of PAF. Platelets have also been shown to significantly contribute to endothelial dysfunction by production of interleukin‐1β through activation of the NLRP3 inflammasome and also by inducing production of inflammatory cytokines by monocytes. Drugs that block down‐stream immunological mediator pathways such as PAF may also be beneficial in the treatment of severe disease.     

Role of Monocytes in the Pathogenesis of Dengue

Diseases caused by dengue virus (DENV) are a major public health problem worldwide, considered one of the infections with more prevalence in tropical and subtropical zones of the world. Despite the intense research in the pathogenesis of DENV, this feature is not well understood. One of the main target cells for DENV infection is monocytes; these phagocytes can play a dual role, since they are essential to control viremia, but they also participate in the induction of tissue damage during DENV infection. Monocytes produce different pro-inflammatory cytokines and chemokines in response to infection, and also mediate endothelial damage. In peripheral blood, monocytes can be divided into three different subpopulations, namely classical, intermediate and non-classical, which differ in frequency, cytokine production, among others. Studies in the last years suggest that non-classical monocytes have higher affinity for microvasculature endothelium compared to other type of monocytes, which implies that they could be more involved in the increase of endothelial permeability observed during DENV infection. This review provides a general view of the role of monocytes and their subpopulations in DENV pathogenesis and its effect in viral replication. Finally, the potential contribution of these phagocytes in the alterations of endothelial permeability is discussed.

     

Differential regulation of angiopoietin 1 and angiopoietin 2 during dengue virus infection of human umbilical vein endothelial cells: implications for endothelial hyperpermeability

Infection with dengue virus (DV) can result in dengue hemorrhagic fever and dengue shock syndrome, where patients suffer from bleeding and plasma leakage involving endothelial cells. Angiopoietins (Ang) 1 and 2 are important angiogenic factors that affect endothelial barrier integrity. In this study, DV was observed to induce endothelial leakage at multiplicity of infection of 10 in primary human umbilical vein endothelial cells (HUVEC) with interendothelial gap formation. Immunostaining of vascular endothelial cadherin (VE-cadherin) and zona occludin 1 (ZO-1) showed the absence of these endothelial junctional proteins at the cell–cell contact zones between adjacent cells. In addition, Ang1 that is required for protecting against endothelial hyperpermeability was found to be down-regulated during DV infection. Treatment with increasing concentrations of recombinant Ang1 was shown to prevent DV-induced endothelial hyperpermeability in a dose-dependent manner by preventing the down-regulation of VE-cadherin and ZO-1 at cell membrane. In contrast, the expression of Ang2, the natural antagonist of Ang1, was observed to be up-regulated during DV infection. Recombinant Ang2 added to HUVEC at non-toxic concentrations showed decreased in transendothelial electrical resistance reading and the down-regulation of VE-cadherin and ZO-1. These findings suggest that DV reduces the expression of Ang1 and enhances the expression of Ang2 in endothelial cells and that this imbalance of Ang 1 and Ang 2 may play a contributing role to the increased permeability of human primary endothelial cells during DV infection.     

Dengue Virus Pathogenesis: an Integrated View

Summary: Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.     

Supernatants from dengue virus type-2 infected macrophages induce permeability changes in endothelial cell monolayers

The ability of dengue virus-infected human monocyte-derived macrophages to induce permeability changes in primary human umbilical vein endothelial cells was investigated. Supernatants from dengue virus type 2-infected monocyte-derived macrophages increased permeability in human umbilical vein endothelial cell monolayers without inducing endothelial cell infection. Production of permeabilising activity from monocyte-derived macrophages occurred after the peak of progeny virus release. TNF-alpha, a known inducer of endothelial cell permeability, was released from dengue virus infected monocyte-derived macrophages but its release did not coincide with release of endothelial cell permeabilising activity. Permeability induction was enhanced by pre-incubation with supernatants from infected monocyte-derived macrophages harvested at the time of peak release of TNF-alpha and infectious virus. Thus, supernatants from dengue virus-infected monocyte-derived macrophages contain factors that increase human umbilical vein endothelial cell permeability, but this is not accompanied by endothelial cell infection or directly correlated with release of dengue virus or TNF-alpha from monocyte-derived macrophages. This model system can be used for further in vitro analysis of mechanisms that may relate to capillary leakage and the development of dengue haemorrhagic fever/dengue shock syndrome.     

FasL/Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype‐2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype‐2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1–2 were constantly very low, whereas Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors 2–4 decreased after dengue virus serotype‐2 infection. This result suggested that dengue virus serotype‐2 may inhibit Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors‐induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype‐2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus‐induced apoptosis of vascular endothelial cells in vitro. J. Med. Virol. 82:1392–1399, 2010. © 2010 Wiley‐Liss, Inc.     

Differential regulation of toll-like receptor-2, toll-like receptor-4, CD16 and human leucocyte antigen-DR on peripheral blood monocytes during mild and severe dengue fever

Dengue fever (DF), a public health problem in tropical countries, may present severe clinical manifestations as result of increased vascular permeability and coagulation disorders. Dengue virus (DENV), detected in peripheral monocytes during acute disease and in in vitro infection, leads to cytokine production, indicating that virus–target cell interactions are relevant to pathogenesis. Here we investigated the in vitro and in vivo activation of human peripheral monocytes after DENV infection. The numbers of CD14⁺ monocytes expressing the adhesion molecule intercellular adhesion molecule 1 (ICAM‐1) were significantly increased during acute DF. A reduced number of CD14⁺ human leucocyte antigen (HLA)‐DR⁺ monocytes was observed in patients with severe dengue when compared to those with mild dengue and controls; CD14⁺ monocytes expressing toll‐like receptor (TLR)2 and TLR4 were increased in peripheral blood from dengue patients with mild disease, but in vitro DENV‐2 infection up‐regulated only TLR2. Increased numbers of CD14⁺ CD16⁺ activated monocytes were found after in vitro and in vivo DENV‐2 infection. The CD14^high CD16⁺ monocyte subset was significantly expanded in mild dengue, but not in severe dengue. Increased plasma levels of tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and interleukin (IL)‐18 in dengue patients were inversely associated with CD14^high CD16⁺, indicating that these cells might be involved in controlling exacerbated inflammatory responses, probably by IL‐10 production. We showed here, for the first time, phenotypic changes on peripheral monocytes that were characteristic of cell activation. A sequential monocyte‐activation model is proposed in which DENV infection triggers TLR2/4 expression and inflammatory cytokine production, leading eventually to haemorrhagic manifestations, thrombocytopenia, coagulation disorders, plasmatic leakage and shock development, but may also produce factors that act in order to control both intense immunoactivation and virus replication.     

Distinct molecular composition of blood and lymphatic vascular endothelial cell junctions establishes specific functional barriers within the peripheral lymph node

Lymph nodes are strategically localized at the interfaces between the blood and lymphatic vascular system, delivering immune cells and antigens to the lymph node. As cellular junctions of endothelial cells actively regulate vascular permeability and cell traffic, we have investigated their molecular composition by performing an extensive immunofluorescence study for adherens and tight junction molecules, including vascular endothelium (VE)-cadherin, the vascular claudins 1, 3, 5 and 12, occludin, members of the junctional adhesion molecule family plus endothelial cell-selective adhesion molecule (ESAM)-1, platelet endothelial cell adhesion molecule-1, ZO-1 and ZO-2. We found that junctions of high endothelial venules (HEV), which serve as entry site for naive lymphocytes, are unique due to their lack of the endothelial cell-specific claudin-5. LYVE-1(+) sinus-lining endothelial cells form a diffusion barrier for soluble molecules that arrive at the afferent lymph and use claudin-5 and ESAM-1 to establish characteristic tight junctions. Analysis of the spatial relationship between the different vascular compartments revealed that HEV extend beyond the paracortex into the medullary sinuses, where they are protected from direct contact with the lymph by sinus-lining endothelial cells. The specific molecular architecture of cellular junctions present in blood and lymphatic vessel endothelium in peripheral lymph nodes establishes distinct barriers controlling the distribution of antigens and immune cells within this tissue.