Subcapsular Sinus Macrophages Research Articles

Fusion Proteins for Versatile Antigen Targeting to Cell Surface Receptors Reveal Differential Capacity to Prime Immune Responses

Targeting of proteins to APCs is an attractive strategy for eliciting adaptive immune responses. However, the relationship between the choice of the targeted receptor and the quality and quantity of responses remains poorly understood. We describe a strategy for expression of Ags including hydrophobic proteins as soluble fusion proteins that are optimized for proteasome-dependent MHC class I-restricted cross-presentation and form stable complexes with a wide variety of targeting Abs. Upon s.c. immunization, these complexes were initially taken up by CD169+ lymph node subcapsular sinus macrophages. In the OVA model system, receptor-targeted antigenic complexes primed specific T and B cell responses in vitro and in vivo at least 100-fold more efficiently than Ag alone. Comparison of 10 targeting receptors allowed us to establish a ranking with respect to priming of CD8+ T cell responses and demonstrated striking differences with respect to the relative efficacy of CD8+ and CD4+ T cell subset and B cell priming. The described fusion proteins should help in developing optimized strategies for targeted delivery of protein Ags in the context of tolerization or vaccination.

Visualizing influenza virus capture in the lymph node following vaccination

Visualizing influenza virus capture in the lymph node following vaccination

Subcapsular sinus macrophages prevent CNS invasion on peripheral infection with a neurotropic virus

Lymph nodes (LNs) capture microorganisms that breach the body’s external barriers and enter draining lymphatics, limiting the systemic spread of pathogens1. Recent work has shown that CD11b+CD169+ macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for clearance of viruses from the lymph and for initiating antiviral humoral immune responses2,3,4. Using vesicular stomatitis virus (VSV), a relative of rabies virus transmitted by insect bites, we show here that SCS macrophages perform a third vital function: they prevent lymph-borne neurotropic viruses from infecting the CNS. Upon local depletion of LN macrophages, ~60% of mice developed ascending paralysis and died 7–10 days after subcutaneous infection with a small dose of VSV, while macrophage-sufficient animals remained asymptomatic and cleared the virus. VSV gained access to the nervous system via peripheral nerves in macrophage-depleted LNs. In contrast, within macrophage-sufficient LNs VSV replicated preferentially within SCS macrophages but not in adjacent nerves. Removal of SCS macrophages did not compromise adaptive immune responses against VSV, but reduced type I interferon (IFN-I) production within infected LNs. VSV-infected macrophages recruited IFN-I producing plasmacytoid dendritic cells to the SCS and additionally were a major source of IFN-I themselves. Experiments in bone marrow chimeric mice revealed that IFN-I must act on both hematopoietic and stromal compartments, including the intranodal nerves, to prevent lethal VSV infection. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral neuroinvasion upon peripheral infection.

A Protease-Dependent Mechanism for Initiating T-Dependent B Cell Responses to Large Particulate Antigens

Ab production is critical for antimicrobial immunity, and the initial step in this process is the binding of Ag to the BCR. It has been shown that small soluble proteins can directly access the lymph node follicles to reach naive B cells, but virus particles must be translocated into follicles via subcapsular sinus macrophages. In this article, we explore how large particulate Ags generate humoral immune responses. Ag-specific follicular B cells rapidly acquired Ag, presented peptide:MHC class II ligands, and produced T-dependent Ab responses following s.c. injection of 1-mum, Ag-linked microspheres, despite the microspheres being confined to the subcapsular sinus. The mechanism of Ag acquisition did not require dendritic cells, subcapsular sinus macrophages, or B cell movement to the subcapsular sinus. Rather, B cell Ag acquisition was protease-dependent, suggesting that some protein Ags are cleaved from the surface of particles to directly initiate humoral immune responses.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

Lymph Node Subcapsular Sinus Macrophages Confer Resistance to CNS Invasion upon Peripheral Infection with a Neurotropic Virus

Lymph nodes (LNs) capture microorganisms that breach the body's external barriers and enter draining lymphatics, thereby limiting the systemic spread of such pathogens1. Recent work has shown that CD11b+CD169+ macrophages, which populate the subcapsular sinus (SCS) of LNs, are critical for clearance of viruses from the lymph and for initiating anti-viral humoral immune responses2,3,4. Using vesicular stomatitis virus (VSV), a relative to rabies virus that is typically transmitted by insect bites, we show here that SCS macrophages perform a third vital function; they prevent lymph-borne neurotropic viruses from infecting the CNS. Upon local depletion of SCS macrophages, ~60% of mice developed ascending paralysis and died within 7-10 days after subcutaneous infection with a small dose of VSV that was readily cleared by macrophage-sufficient control animals. VSV gained access to the nervous system via peripheral nerves in macrophage-depleted LNs. In contrast, in macrophage-sufficient LNs VSV replicated preferentially within SCS macrophages but failed to replicate in adjacent nerves. Removal of SCS macrophages did not compromise the humoral or T cell responses against VSV, but it dramatically reduced the production of type I interferon (IFN-I) within infected LNs. VSV-infected macrophages recruited IFN-I producing plasmacytoid dendritic cells to the SCS and additionally were a major source of IFN-I themselves. Experiments in bone marrow chimeric mice lacking the IFN-I receptor in either hematopoietic or stromal cells revealed that IFN-I must act on both compartments, including the intranodal nerves, to prevent lethal VSV infection. These results identify SCS macrophages as crucial gatekeepers to the CNS that prevent fatal viral neuroinvasion upon peripheral infection.

The missing link in the affinity maturation chain

Knowledge of the B cell's capacity to recognize native antigen has diluted recognition of the importance of B cell interaction and/or communication with accessory cells to initiate the humoral immune response. In addition, the mechanisms by which antigens gain access to the follicle, and thus to the B cell repertoire, were largely unknown until recently. In the July issue of Nature Immunology, Phan et al.1 extensively characterize the macrophage population located at the follicle junction with the lymph node subcapsular sinus (SCS). The authors show an important role for immune complex relay by SCS macrophages and non-cognate B cells in driving antibody affinity maturation at the germinal centre (GC).

Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation.

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-α1β2 and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, non-cognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular sinus to the germinal center and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

Subcapsular sinus macrophages prevent lethal vesicular stomatitis virus (VSV) infection (43.11)

Abstract When a pathogen escapes innate host responses at the site of entry, it has the potential to spread systemically by accessing tissue-draining lymph vessels that channel interstitial fluid to the venous circulation. Before reaching the blood, the tissue-derived lymph must first pass through draining lymph nodes (LNs). LN act as filter stations preventing pathogens from reaching the systemic circulation and they are the staging grounds for adaptive immune responses, providing a specialized environment for the presentation of antigens to B and T cells. It is unclear how viruses are cleared from afferent lymph and how antiviral immune responses are generated. We have previously identified a population of CD11b+CD169+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of LNs that capture viral particles within minutes after subcutaneous (sc) injection. Using VSV, an acutely cytopathic RNA virus closely related to rabies virus, we show here that these macrophages are critical to prevent viral access to nerve termini located in the SCS. Indeed, mice depleted of SCS macrophages develop an ascending CNS paralysis that leads to death in 50% of the animals 6-10 days after sc infection. Macrophage-depleted mice die despite mounting an increased neutralizing antibody (nAb) response. The notion that nAb are dispensable for protection against a sc infection (the natural route of transmission of this virus) is further demonstrated by the fact that antibody-deficient mice, which die upon intravenous infection, survive a sc VSV infection as long as SCS macrophages are present. While nAb are unnecessary for survival, B cells are required, and their protective role presumably relies on maintenance of LN architecture and normal macrophage phenotype.

Conduits Mediate Transport of Low-Molecular-Weight Antigen to Lymph Node Follicles

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.

Murine cytomegalovirus displays selective infection of cells within hours after systemic administration

A distinctive feature of the cytomegaloviruses is their wide tissue tropism, demonstrated by the infection of many organs and cell types in an active infection. However, in experimental models of systemic infection, the earliest stages of infection are not well characterized, and it is unclear whether only certain cells are initially infected. Using a recombinant murine cytomegalovirus (MCMV) expressing green fluorescent protein (GFP), we tracked viral infection after systemic administration via intraperitoneal injection and showed that specific cells are infected within the first hours. We provide evidence that MCMV traffics as free virus from the peritoneal cavity into the mediastinal lymphatics, providing access to the bloodstream. We demonstrate that MCMV productively infected CD169(+) subcapsular sinus macrophages in the mediastinal lymph nodes, ER-TR7(+) CD29(+) reticular fibroblasts in the spleen and hepatocytes. Infection in the spleen followed a distinctive pattern, beginning in the marginal zone at 6 h and spreading into the red pulp by 17 h. By 48 h after infection, there was widespread infection in the spleen and liver with degeneration of infected cells. In addition, infected dendritic cells appeared in the white pulp of the spleen at 48 h post-infection. On the other hand, cowpox virus showed a different pattern of infectivity in the spleen and liver. Thus, early MCMV infection produces a distinct pattern of infection of selective cells.

Surface antigens are rapidly separated from bacterium‐sized microspheres in the subcapsular sinus and acquired by antigen‐specific follicular B cells

Antibody production is critical for antimicrobial immunity. The initial step in this process is the binding of antigen to the B cell receptor. Yet, the size of many microbes prevents them from entering the lymph node follicles where naïve B cells reside. Here, we show that antigen‐specific follicular B cells rapidly acquired antigen, produced peptide:MHC II complexes, and secreted antibody in a T cell‐dependent fashion following subcutaneous injection of bacterium‐sized antigen‐coupled particles even though the particles were confined to the subcapsular sinus. This form of B cell antigen acquisition did not depend on dendritic cells or subcapsular sinus macrophages. These results suggest that proteins released from the surface of microbes directly access the follicles to initiate the humoral response.

Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells

Lymph nodes prevent the systemic dissemination of pathogens such as viruses that infect peripheral tissues after penetrating the body's surface barriers. They are also the staging ground of adaptive immune responses to pathogen-derived antigens. It is unclear how virus particles are cleared from afferent lymph and presented to cognate B cells to induce antibody responses. Here we identify a population of CD11b+CD169+MHCII+ macrophages on the floor of the subcapsular sinus (SCS) and in the medulla of lymph nodes that capture viral particles within minutes after subcutaneous injection. Macrophages in the SCS translocated surface-bound viral particles across the SCS floor and presented them to migrating B cells in the underlying follicles. Selective depletion of these macrophages compromised local viral retention, exacerbated viraemia of the host, and impaired local B-cell activation. These findings indicate that CD169+ macrophages have a dual physiological function. They act as innate 'flypaper' by preventing the systemic spread of lymph-borne pathogens and as critical gatekeepers at the lymph-tissue interface that facilitate the recognition of particulate antigens by B cells and initiate humoral immune responses.

Potential role of the mannose receptor in antigen transport

Potential endogenous ligands for the cysteine rich domain of the murine mannose receptor (MR) have been detected in marginal zone metallophilic macrophages in spleen and subcapsular sinus macrophages in lymph nodes of naive mice by immunohistochemistry using a Fc chimeric protein. Additional labelling was observed in follicular dendritic cells and migratory dendritic cells in immunised animals. Based on this labelling pattern and the identification of a soluble form of the MR in macrophage-conditioned media and mouse serum, we propose a novel role for this receptor in antigen transport.

Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse.

We used a panel of monoclonal antibodies and immunocytochemistry to identify macrophages and dendritic cells in mice that are deficient in macrophage colony stimulating factor (M-CSF or CSF-1) because of the recessive osteopetrotic (op/op) mutation. Prior work had shown that osteopetrosis is associated with a lack of osteoclasts, phagocytic cells required for remodelling in bone. Additional macrophage populations proved to be very M-CSF dependent. op/op mice had few and sometimes no peritoneal cavity phagocytes, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages. Other populations, however, reached substantial levels in the absence of M-CSF, including phagocytes in the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, liver (Kupffer cells), lung (alveolar macrophages) and brain (microglia). Dendritic cells, which are specialized accessory cells for T-dependent immune responses and tolerance, were readily identified in skin and in the T-dependent regions of spleen, lymph node and Peyer's patch. The identification of dendritic cells utilized antibodies to MHC class II products and four different antigens that are primarily expressed by these accessory cells. Our findings indicate that only a few macrophage populations are critically dependent upon M-CSF in vivo. With respect to dendritic cells, the data are consistent with prior in vitro work where it was noted that GM-CSF but not M-CSF supported dendritic cell viability, function and growth.

The influence of afferent lymphatic vessel interruption on vascular addressin expression.

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.

The Functional Activity of High Endothelial Venules: A Role for the Subcapsular Sinus Macrophages in the Lymph NodeREIN

Adherence of lymphocytes to high endothelial venules (HEV 3) is the first step in normal lymphocyte emigration and recirculation.At sites of chronic inflammation, venules often become high-walled and may also be a site for leukocytes to leave the bloodstream.The immunologic and inflammatory mediators, responsible for these effects on endothelial cells, may be important for the maintenance and function of HEV in physiological conditions. It is reported here that the morphological and functional aspects of HEV can be studied by organ cultures of lymph nodes (LN).At 24 h of culture, the appearance of the node was still quite normal, whereas the HEV became flat-walled, with a 45–50 % reduction in the capacity to bind lymphocytes.This decrease in function of HEV could be reduced when LN were cultured in the presence of lipopolysaccharides (LPS).The effect of LPS on the function of HEV was presumably mediated by macrophages in the subcapsular sinus, because HEV in LN, which were depleted of subcapsular sinus and medullary macrophages previous to culture, could not be stimulated by addition of LPS to the cultures.

Macrophages and the activity of high endothelial venules. The effect of interferon‐γ

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3-reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon-gamma (rIFN-gamma). Injection of rIFN-gamma directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN-gamma could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.