Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells

The mechanism of B cell-antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor-dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.     

Transport of immune complexes from the subcapsular sinus into the lymph node follicles of the rat

To study the mode of transport of immune complexes from the subcapsular sinus into the follicles of draining popliteal lymph nodes, horse radish peroxidase (HRP)-anti HRP was injected in rat footpads. Within six minutes, complexes were already present in the subcapsular sinus freely or attached to the plasma membrane of different types of cell including cells forming the stroma. A few minutes later, complexes were also seen in the deeper part of the outer cortex, and after two hours they had reached the periphery of the follicles. They were always seen scattered between lymphoid and non-lymphoid cells. After one day, complexes were present on well-developed follicular dendritic cells. After injection of HRP, no localization of this antigen was observed in the deeper part of the outer cortex including the follicles. These results strongly suggest that HRP-anti HRP complexes are passively transported through the outer cortex into the follicles where they are trapped and retained by follicular dendritic cells.     

Structural and functional changes to lymph nodes in ageing mice

Lymph nodes (LN) are secondary lymphoid organs spread throughout the lymphatic system. They function to filter pathogenic material from the lymphatic fluid to maintain the health of the organism. Subcapsular sinus macrophages (SCSM) are among the first‐responders within the LN due to their strategic location within the subcapsular sinus region. These macrophages aid the delivery of immune complexes to B cells and follicular dendritic cells (FDC) within the LN. Here we show an increase in SCSM and other macrophage populations within aged LN. However, immune complex uptake by macrophages within LN was not altered with age, nor was immune complex uptake by B cells. LN stromal cell populations, important in immune responses and the localization and survival of leucocytes, were altered in their representation and distribution in aged LN. In particular, FDC regions were decreased in size and had decreased chemokine CXCL13 expression. Furthermore, the retention of immune complexes by FDC was decreased in aged LN at 24 hr post‐injection. As FDC are important in the maintenance of germinal centre responses, the decreased retention of immune complex in aged LN may contribute to the reduced germinal centre responses observed in aged mice.     

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.     

Prolonged antigen half-life in the lymphoid follicles of specifically immunized mice

The kinetics of clearance of ¹²⁵I from the popliteal lymph nodes and feet of human serum albumin (HSA)-immunized mice was studied following the injection of [¹²⁵I]-HSA into the hind footpads. Antigen was cleared from both locations rapidly for the first few days. The antigen half-life (T½) during this period was only a matter of hours. By the end of the first week, however, the rate of clearance in both sites had changed markedly. The antigen T½ in the node between the first and sixth week was 8.1 weeks (95% confidence interval between 5.1 and 20 weeks) and the antigen T½ in the foot was 6.1 weeks (95% confidence interval between 3.7 and 16.6 weeks). There was, however, about twenty times more radioactivity in the feet than in the popliteal nodes. Autoradiography of popliteal lymph nodes revealed that initially antigen was trapped in the medulla, subcapsular sinus, superficial cortex and around lymphoid follicles. During the first few days antigen was cleared from all sites except the follicles. The radioactivity initially trapped in the medulla, subcapsular sinus, and superficial cortex appeared to have been associated with macrophages. Studies with peritoneal macrophages indicated an antigen T½ in these cells of 2 h (95% confidence interval between 1.5 and 3 h). The initial rapid clearance of antigen trapped and catabolized by macrophages and the long-term retention of antigen in the follicles is probably attributable to trapping and retention by follicular dendritic cells. The large pool of antigen trapped in the foot did not appear to serve as a depot to replace antigen degraded in the node, since amputation of the foot did not alter the level of antigen retained in the node. The long antigen T½ in the lymph node follicles indicates that antigen is available in the lymph node to play a role in the maintenance and regulation of immune responses for many months or even years.     

Complement receptors CD21 and CD35 in humoral immunity

Summary:  The complement system is a family of proteins that is involved in both innate and adaptive immunity. Complement receptors CD21 and CD35, which recognize activated products of C3 and C4, are predominantly expressed on B cells and follicular dendritic cells (FDCs) in the mouse. In this review, we focus on the role of FDC-expressed CD21 and CD35 in humoral immunity. They are the principle receptors for uptake and retention of immune complexes. In their absence, memory B-cell survival is markedly impaired. This is likely because of the lack of antigen but could also reflect a role for complement C3d ligand. How antigen is transported to FDCs remains an open question. In recent unpublished work using multiphoton intravital imaging, we found that small protein antigens presented in the lymph drain rapidly into B-cell follicles and are taken up by FDCs in a complement-dependent manner.     

Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non-lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM-1). In this report we investigated the appearance and follicular tissue distribution of VCAM-1 in murine peripheral lymphoid tissues, and compared VCAM-1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM-1 with other murine FDC-associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC-M1) revealed that the display of VCAM-1 is restricted to a subset of CR1.2-positive FDCs. We found that the expression of VCAM-1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM-1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM-1), with some slight differences in occurrence. The appearance of VCAM-1 and MAdCAM-1 antigens on FDCs may serve as indicators of FDC activation.     

The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles

The initial step in a humoral immune response involves the acquisition of antigens by B cells via surface immunoglobulin. Surprisingly, anatomic studies indicate that lymph-borne proteins do not have access to the follicles where naive B cells reside. Thus, it is unclear how B cells acquire antigens that drain to lymph nodes. By tracking a fluorescent antigen and a peptide:MHC II complex derived from it, we show that antigen-specific B cells residing in the follicles acquire antigen within minutes of injection, first in the region closest to the subcapsular sinus where lymph enters the lymph node. Antigen acquisition, presentation, and subsequent T cell-dependent activation did not require B cell migration through the T cell area or exposure to dendritic cells. These results indicate that the humoral response is initiated as soluble antigens diffuse directly from lymph in the subcapsular sinus to be acquired by antigen-specific B cells in the underlying follicles.     

Appearance and phenotype of murine follicular dendritic cells expressing VCAM‐1

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non‐lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM‐1). In this report we investigated the appearance and follicular tissue distribution of VCAM‐1 in murine peripheral lymphoid tissues, and compared VCAM‐1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM‐1 with other murine FDC‐associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC‐M1) revealed that the display of VCAM‐1 is restricted to a subset of CR1.2‐positive FDCs. We found that the expression of VCAM‐1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM‐1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM‐1), with some slight differences in occurrence. The appearance of VCAM‐1 and MAdCAM‐1 antigens on FDCs may serve as indicators of FDC activation. Anat Rec 268:160–168, 2002. © 2002 Wiley‐Liss, Inc.     

Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC) with emphasis on the ultrastructural demonstration of Ia antigens

The antigenic phenotype of mouse lymph node follicular dendritic cells (FDCs) was studied by immunocytochemical techniques. Indirect fluorescence was used in conjunction with monoclonal antibodies to localize FDC surface antigens on FDC-enriched cell preparations and in cryostat sections. Lymph nodes from rats and mice were also labeled directly for Ia antigens with fluorescein- or peroxidase-conjugated Ia-specific monoclonal antibodies (i.e., MRC Ox4 and 10-2.16, respectively). Lymphoid tissue was also prepared for electron microscopy to allow clear distinction between Ia antigens of B lymphocytes and FDCs in situ. In these experiments, gold-labeled antigen was used to clearly identify FDCs and their processes among the Ia-positive cells of lymph node follicles. The labeling observed by light and electron microscopy showed that FDCs expressed Ia in situ and in vitro. Additional surface determinants shown to be expressed by FDCs included H2-K, common leukocyte antigen, and the receptor for the Fc portion of IgG1 and IgG2b. Neither macrophage antigens, such as Mac-1, Mac-2, Mac-3, and F4/80, nor the lymphocyte markers Ly-1, Ly-2, and Thy-1 were expressed by FDCs. Thus, the antigenic phenotype of FDCs, along with their distinctive dendritic morphology, their nonphagocytic and nonadherent nature, and their ability to trap and retain immune complexes on their plasma membrane, identifies them as a unique cell population.     

Stromal cell organisation in the mouse lymph node. A light and electron microscopic investigation using the zinc iodide-osmium technique

The organisation of the stromal cell compartment in the mouse lymph node was studied by light and electron microscopy after tissue impregnation by the zinc iodide-osmium (ZIO) method. Fibroblastic reticular cells (FRCs) represented the main stromal cell population. These cells were located both in the cortical region and in the medulla and exhibited various configurations. In the cortex, FRCs were fusiform in shape and came into close proximity with the floor of the subcapsular sinus. In the medulla, the FRCs were shaped like irregular dendritic cells which formed a complex 3-dimensional network. The FRCs surrounded vascular structures such as capillaries and/or high endothelial venules; in these instances they were organised in a discontinuous sheath-like fashion around the vessel wall. By light and electron microscopy, FRCs have been observed to come in close spatial relationship with a number of cells in the lymph node, including sinus endothelial cells, the endothelium of high endothelial venules and capillaries, various types of lymphocytes, follicular dendritic cells and interdigitating cells. These microanatomical features are consistent with the proposal that FRCs may be involved in the communicative networks between the different lymph node compartments. In particular, the FRCs may be involved in the transport of molecules from the sinus compartment to the high endothelial venules or to the distinct cell populations in the lymphoid parenchyma.     

The effect of age on antigen retention in lymphoid follicles and in collagenous tissue of mice

Our major objectives were to determine (1) when mice develop the cellular mechanisms necessary to trap and retain immune complexes in lymphoid follicles, (2) whether the ability to trap and retain immune complexes in lymphoid and collagenous tissues was maintained in old animals, and (3) whether the pattern of antigen localization in lymphoid follicles was altered as a consequence of ageing. Mice were passively immunized with a standardized amount of specific antibody and then challenged in the foot pads with radioactive antigen. The results indicated that newborn, and 1- and 2-week-old BALB/c mice lacked the cells or cellular mechanism necessary for trapping, localizing, and retaining immune complexes in lymphoid follicles. By 3 weeks, however, this ability to trap, retain, and localize antigen became apparent. The ability to trap and retain immune complexes on tendons and in lymphoid tissues was maintained as long as 27-30 months. A comparison of the quantity of antigen retained per mg of tissue in young-adult (6 months of age) and in old (27-30 months of age) mice indicated that old mice retained slightly more antigen in lymphoid tissues and substantially more on tendons. Antigen retained in spleens of both young-adult and old mice was localized in follicles by 24 h. However, the antigen retained in lymph nodes of old mice remained in the subcapsular sinus and adjacent superficial cortex and was not localized in follicles even after a full week. The cellular mechanisms necessary to trap and retain immune complexes in lymphoid tissue appear to develop in BALB/c mice a few weeks after birth and persist throughout life. However, localization of antigen in the lymphoid follicles of lymph nodes diminishes in old mice. The antigen trapping capability of collagenous tissues was not only maintained in old mice but was substantially increased.     

FCA和FICA可增强淋巴滤泡捕捉抗原的能力

目的　探讨向体内注入非抗原类物质能否引起反应性淋巴滤泡形成 ,观察体内注入FCA(完全弗式佐剂 )、FICA(不完全弗式佐剂 )对滤泡树突状细胞 (FDC)捕捉抗原的能力有无影响。方法　将FCA、FI CA分别注入小鼠足底 ,数日后取出腘淋巴结 ,应用PNA B法及PAP免疫组化法染色 ,采取三维重塑方法 ,统计淋巴滤泡、FDC细胞群数量。结果　注入FCA、FICA后第 5日出现初级淋巴滤泡 ,第 3 5日数量达到高峰 ,之后逐渐减退。同时 ,滤泡树突状细胞 (FDC)与B细胞聚集同步出现。结论　动物体内注入有丝分裂物质FCA、FICA引起引流淋巴结内次级淋巴滤泡形成 ,初级滤泡形成过程中B细胞聚集与FDC形成是同步发生的     

Anamnestic responses induced by antigen persisting on follicular dendritic cells from cyclophosphamide-treated mice

In contrast to most mouse lymph node cells, follicular dendritic cells (FDCs) resist cyclophosphamide (Cy; 300 mg/kg)-mediated destruction in vivo. In this study we sought to determine if antigen-bearing FDCs from Cy-treated animals maintained biological activity. We were especially interested in whether FDCs from Cy-treated animals could stimulate an antibody response when combined with primed spleen cells and whether the FDCs needed to be intact and viable for stimulation to occur. The effect of Cy treatment on lymph node histology, number of T cells and B cells, and the 'spontaneous antibody response' was determined. Cy treatment resulted in a massive depletion of the lymph node cortex and a loss of follicles and germinal centres. Over 90% of B cells in the lymph node were eliminated. The paracortex was more resistant although nearly 80% of T cells were eliminated. Cy treatment also eliminated the 'spontaneous antibody response' as established by in vitro culture or after adoptive transfer. The addition of primed spleen cells to antigen-bearing FDCs including sonicated non-viable FDCs from Cy-treated animals resulted in an anamnestic antibody response. Memory lymphocytes, injected into the hind foot pads of Cy-treated animals, migrated to the follicular area of popliteal lymph nodes and cells from these reconstituted nodes spontaneously responded upon subsequent adoptive transfer. It was concluded that antigen retained on Cy-treated FDCs maintains its immunogenicity and is capable of inducing a 'spontaneous antibody response' or an anamnestic response. Furthermore antigen on FDCs or on fragments of FDCs from one animal can interact with memory cells from another animal to induce a productive antibody response. Lymph nodes enriched for FDCs by Cy treatment should be a good source of FDCs for isolation and further study of the nature of this interaction.     

Mechanism of follicular trapping: double immunocytochemical evidence for a contribution of locally produced antibodies in follicular trapping of immune complexes.

Using two different antigen-enzyme conjugates and a double immunocytochemical staining technique, we demonstrate the localization patterns of two different specific antibodies in the same spleen section. During the early immune responses against simultaneously injected human gamma globulin (HGG), and bovine gamma globulin (BGG) in rabbits, the localization patterns of extracellular anti-HGG antibodies and extracellular anti-BGG antibodies in the follicles overlap only partly. It was shown in earlier studies that extracellular antibodies trapped in the follicles represent antigen-antibody complexes having free binding sites for the antigen. The fact that localization patterns do not overlap extensively, whereas it has been shown in earlier studies that follicular dendritic cells (FDCs) show no specificity with respect to the immune complexes to be captured, leads to the following conclusion. After formation of immune complexes from antibody molecules released by specific antibody-forming cells in the follicles and antigen present in excess between the cells, part of these complexes are trapped by adjacent FDCs. Results are discussed with respect to the possible role of follicular immune complexes in the generation of immunological memory.     

Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells

Secreted IgM and complement are important mediators in the optimal initiation of primary T-dependent humoral immune responses. Secreted IgM serves as a natural adjuvant by enhancing the immunogenicity of protein antigens, perhaps as a result of IgM's ability to facilitate antigen deposition onto follicular dendritic cells (FDCs) and promote rapid germinal center (GC) formation. To understand how IgM enhances adaptive immune responses, we investigated the mechanism by which IgM-containing immune complexes (IgM-IC) are transported to FDCs as a first step in GC formation. We demonstrate that IgM-IC localize first to the splenic marginal zone (MZ) where the IgM-IC bind MZ B cells in a complement and complement receptor (CR1/2) dependent process. MZ B cells then transport the IgM-IC into the follicle for deposition onto FDCs. Mice with reduced numbers of MZ B cells trap IgM-IC on FDC less efficiently, whereas mice with reduced numbers of follicular B cells trap IgM-IC normally. The functional elimination of MZ B cells abrogates the ability of FDCs to trap IgM-IC. Transfer of B cells with associated IgM-IC into naive mice results in deposition of IgM-IC onto FDC by MZ B cells. The results demonstrate an IgM and complement-dependent role for MZ B cells in the fate of antigen early in the initial phases of T-dependent immune responses. The data also establish an important role for CR1/2 on MZ B cells in the efficient binding and transport of IgM-IC to FDCs, which we suggest is an important first step in initiating adaptive immune responses.     

滤泡树突状细胞与生发中心反应研究的新进展

生发中心是在T细胞依赖性抗体应答过程中于外周淋巴组织内形成的一个特殊的结构。在GC内,受抗原刺激而活化的B细胞进行克隆扩增、IgV区基因的体细胞高度突变、亲和力成熟以及同类型转换,最终形成记忆性B细胞或是产生Ig的浆细胞。在GC内B细胞增殖的同时,也启动了凋亡机制,以确保最终形成的记忆B细胞或浆细胞对抗原的高度特异性。FDCs是参与再次免疫应答的重要细胞,它主要是通过表面的FcR和CR将免疫复合物结合在细胞膜上,并选择性的将抗原递呈给表达高亲和力BCR的B细胞,使之激活并产生抗体或形成记忆B细胞。因此,FDCs在生发中心反应、免疫记忆的维持、B细胞的分化、成熟以及记忆B细胞的形成具有极其重要的作用。但最近的研究对FDCs及其结合的免疫复合物的重要性提出了质疑,认为FDCs在生发中心反应、B细胞的分化、成熟以及记忆B细胞的形成中的作用很可能是非特异性的,并对驻留在FDCs表面的免疫复合物的其它潜在功能进行了讨论。     

Antigen archiving by lymph node stroma: A novel function for the lymphatic endothelium

Secondary lymphoid stroma performs far more functions than simple structural support for lymphoid tissues, providing a host of soluble and membrane‐bound cues to trafficking leukocytes during inflammation and homeostasis. More recently it has become clear that stromal cells can manipulate T‐cell responses, either through direct antigen‐mediated stimulation of T cells or more indirectly through the retention and management of antigen after viral infection or vaccination. In light of recent data, this review provides an overview of stromal cell subsets and functions during the progression of an adaptive immune response with particular emphasis on antigen capture and retention by follicular dendritic cells as well as the recently described “antigen archiving” function of lymphatic endothelial cells (LECs). Given its impact on the maintenance of protective immune memory, we conclude by discussing the most pressing questions pertaining to LEC antigen capture, archiving and exchange with hematopoetically derived antigen‐presenting cells.     

Immunocytochemical study on the immune complex and germinal center of synovial tissue of rheumatoid arthritis with special regard to complement and follicular dendritic cells

To analyze the immunological role of lymphoid germinal centers and follicular dendritic cells (FDC) in synovial tissue of rheumatoid arthritis (RA), we tried to detect the immune complex in germinal centers by light and electron microscopic immunocytochemistry with special emphasis on immunoglobulin, complement components, RA factor, and DRC-1 antigen. Immunoglobulins mainly distributed intercellularly in the germinal center in a lacy network pattern, and show partial intracytoplasmic localization in some germinal center lymphoid cells. Early complement components of classical pathway (C1q, C4, C3c, and C3d) and RA factor distributed lacily similarily to immunoglobulin, but intracytoplasmic positivity is never observed. These coexistent positive sites are identical to DRC-1 positive sites which are the surface of extended processes of FDC membrane. A similar finding is observed in primary follicles or lymphoid aggregates less often than germinal centers. These results indicate that some germinal centers trap the immune complex, including RA factor at least closely related with FDCs, and also RA factor is one of the triggers of antigen as well as developing germinal centers.     

The Localization of Antigen in Lymph Node Follicles of Congenitally Athymic Nude Rats

We have examined the postulated dependence on T cells of follicular retention of antigen by studying antigen retention in the draining lymph nodes of congenitally athymic, nude rats after local injections of horseradish peroxidase (HRP). The lymphoid tissues of these rats contained germinal centres and follicular dendritic cells (FDC) that were ultrastructurally identical to those seen in euthymic rats and expressed the differentiation antigen MRC OX2. Nude rat FDC captured and retained locally injected antigen on their surfaces, but as with euthymic rats, only in the presence of previously injected anti-HRP antibody. This demonstrates that the FDC mature both morphologically and functionally in the absence of a thymus or T cells. However, in contrast to euthymic rats, there was no detectable antigen retention in nude rats that had been actively immunized by repeated intraperitoneal injections with HRP for 3 months. The lower number of germinal centres observed in athymic animals compared with their euthymic littermates could thus be explained by deficient production of specific antibody of the isotype necessary for follicular localization of environmental antigens.