Lymph node stromal cells: subsets and functions in health and disease.

Lymph nodes (LNs) aid the interaction between lymphocytes and antigen-presenting cells, resulting in adequate and prolonged adaptive immune responses. LN stromal cells (LNSCs) are crucially involved in steering adaptive immune responses at different levels. Most knowledge on LNSCs has been obtained from mouse studies, and few studies indicate similarities with their human counterparts. Recent advances in single-cell technologies have revealed significant LNSC heterogeneity among different subsets with potential selective functions in immunity. This review provides an overview of current knowledge of LNSCs based on human and murine studies describing the role of these cells in health and disease.

MAdCAM-1 does not play a central role in the early pathophysiology of autoimmune hepatitis.

INTRODUCTION: CD4+ T cells are thought to have a central role in the pathogenesis of autoimmune hepatitis (AIH). Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs homing of CD4+ T cells in the alimentary tract and is a therapeutic target in inflammatory bowel diseases. Here we assessed MAdCAM-1 expression in AIH and viral hepatitis and related its expression with immune infiltrate analysis and histopathological key features. METHODS: Hepatic portal areas of pretreatment biopsies (n=10) and follow-up biopsies (n=9) of patients with a confirmed diagnosis of AIH were assessed for MAdCAM-1 expression and infiltrate composition using immunohistochemistry and multispectral imaging (Vectra® Polaris™). Controls consisted of biopsies of patients with untreated chronic viral hepatitis B or C (n=22). RESULTS: MAdCAM-1 expression on endothelium was sparsely present in portal fields of two treatment-naïve AIH patients. Three patients showed MAdCAM-1 expression within lymphoid aggregates. No expression of significance (including single-cell expression) was observed in the remaining 6 patients. In contrast, viral hepatitis C biopsies showed endothelial MAdCAM-1 expression in 8 of 13 untreated patients. Densities of both B-cells (CD20+) and CD4+ T-cells (CD3+ CD8-) were increased in AIH and viral hepatitis patients with MAdCAM-1 expression. CONCLUSION: MAdCAM-1 was detected in liver biopsies in a minority of patients with AIH at the time of diagnosis suggesting no central role in its pathophysiology. Lymphoid or reticular MAdCAM-1 pattern expression was associated with more dense infiltrates of both B-cells and CD4+ T-cells, and may be related to the formation of secondary lymphoid follicles.

Identification of a soluble form of a ligand for the lymphocyte homing receptor.

Lymphocytes are engaged in constant trafficking from the blood into secondary lymphoid tissues, such as peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP). The initial step in this process is the binding of lymphocytes to high endothelial venules (HEV), and in the case of trafficking of cells to the PLN, it is required that they bear the L-selectin surface receptor. Using a chimeric protein, combining the extracellular domains of L-selectin with a human immunoglobulin (Ig) G1 Fc region (L-selectin-IgG), we have probed the expression of ligands for this receptor on HEV and in cell lysates. Two sulfated glycoproteins of 50 and 90 kD have been identified in lysates from PLN and MLN, but not PP. Here we show that the 50-kD molecule is secreted in organ cultures in vitro and is present in the blood of normal animals. Indeed, normal serum inhibits lymphocyte binding to HEV by approximately 50% in an in vitro assay. This inhibitory activity can be removed by passage of the serum over an L-selectin-IgG column and has a molecular mass of approximately 50 kD. We speculate on the possible reasons for secretion of a homing receptor ligand.

Surface lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs.

For more than a decade, the biological roles and the apparent redundancy of the cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) have been debated. LT alpha exists in its soluble form as a homotrimer, which like TNF only binds the TNF receptors, TNF-R55 or TNF-R75. The cell surface form of LT exists as a heteromer of LT alpha and LT beta subunits and this complex specifically binds the LT beta receptor (LT beta-R). To discriminate the functions of the LT and TNF systems, soluble LT beta-R-immunoglobulin (Ig) or TNF-R-Ig fusion proteins were introduced into embryonic circulation by injecting pregnant mice. Exposure to LT beta-R-Ig during gestation disrupted lymph node development and splenic architecture in the progeny indicating that both effects are mediated by the surface LT alpha/beta complex. These data are the first to identify a cell surface ligand involved in immune organ morphogenesis. Moreover, they unambiguously discriminate the functions of the various TNF/LT ligands, provide a unique model to study compartmentalization of immune responses and illustrate the generic utility of receptor-Ig fusion proteins for dissecting/ordering ontogenetic events in the absence of genetic modifications.

Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE.

Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/)- mice correlates with a significant reduction in lymphotoxin (LT)alphabeta(+)alpha(4)beta(7)(+)CD45(+)CD4(+)CD3(-) cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/)- mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTalphabeta expression on CD45(+) CD4(+)CD3(-) cells, as LNs could not be induced in LTalpha(-/)- mice. LTalpha(-/)- mice also showed defects in the fate of CD45(+)CD4(+)CD3(-) cells similar to TRANCE(-/)- mice. Thus, we propose that both TRANCE and LTalphabeta regulate the colonization and cluster formation by CD45(+) CD4(+)CD3(-) cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.

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.

Requirement for RORgamma in thymocyte survival and lymphoid organ development.

Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. RORgamma was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3-CD4+CD45+ cells that normally express RORgamma and that are likely early progenitors of lymphoid organs. Hence, RORgamma has critical functions in T cell repertoire selection and lymphoid organogenesis.

Selective modulation of the expression of L-selectin ligands by an immune response.

BACKGROUND: The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L-selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells. The cDNA encoding Sgp50 has been cloned, and its product, which has been designated GlyCAM-1, is secreted. The third ligand, Sgp200, is both secreted and cell-associated. We have investigated how the expression of these sulphated glycoproteins is regulated during an immune response. RESULTS: Here we demonstrated that, during a primary immune response, the expression and secretion of both GlyCAM-1 and Sgp200 are reduced, recovering to normal levels 7-10 days after antigen stimulation. In contrast, the expression of cell-associated CD34 and Sgp200 is relatively unaffected. These results may account for the modest decreases in the binding of an L-selectin-IgG fusion protein to high endothelial venules of inflamed peripheral lymph nodes that have been observed after antigen exposure. In vivo experiments show that, following the decrease in the levels of secreted GlyCAM-1 and Sgp200, migration of lymphocytes from the blood stream into lymph nodes remains L-selectin-dependent, but more lymphocytes home to antigen-primed than unprimed peripheral lymph nodes. CONCLUSIONS: We suggest that the secreted forms of the L-selectin ligands GlyCAM-1 and Sgp200 act as modulators of cell adhesion, and that cell-associated CD34 and Sgp200 are the ligands that mediate the initial loose binding of lymphocytes to high endothelial venules.

The role of cell adhesion molecules in the development of IDDM: implications for pathogenesis and therapy.

IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM.

The functional activity of high endothelial venules: a role for the subcapsular sinus macrophages in the lymph node.

Adherence of lymphocytes to high endothelial venules (HEV3) 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.

The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms.

Mucosal surfaces are constantly exposed to environmental antigens, colonized by commensal organisms and used by pathogens as points of entry. As a result, the immune system has devoted the bulk of its resources to mucosal sites to maintain symbiosis with commensal organisms, prevent pathogen entry, and avoid unnecessary inflammatory responses to innocuous antigens. These functions are facilitated by a variety of mucosal lymphoid organs that develop during embryogenesis in the absence of microbial stimulation as well as ectopic lymphoid tissues that develop in adults following microbial exposure or inflammation. Each of these lymphoid organs samples antigens from different mucosal sites and contributes to immune homeostasis, commensal containment, and immunity to pathogens. Here we discuss the mechanisms, mostly based on mouse studies, that control the development of mucosal lymphoid organs and how the various lymphoid tissues cooperate to maintain the integrity of the mucosal barrier.

Colonic patch and colonic SILT development are independent and differentially regulated events.

Intestinal lymphoid tissues have to simultaneously ensure protection against pathogens and tolerance toward commensals. Despite such vital functions, their development in the colon is poorly understood. Here, we show that the two distinct lymphoid tissues of the colon-colonic patches and colonic solitary intestinal lymphoid tissues (SILTs)-can easily be distinguished based on anatomical location, developmental timeframe, and cellular organization. Furthermore, whereas colonic patch development depended on CXCL13-mediated lymphoid tissue inducer (LTi) cell clustering followed by LTα-mediated consolidation, early LTi clustering at SILT anlagen did not require CXCL13, CCR6, or CXCR3. Subsequent dendritic cell recruitment to and gp38(+)VCAM-1(+) lymphoid stromal cell differentiation within SILTs required LTα; B-cell recruitment and follicular dendritic cell differentiation depended on MyD88-mediated signaling, but not the microflora. In conclusion, our data demonstrate that different mechanisms, mediated mainly by programmed stimuli, induce the formation of distinct colonic lymphoid tissues, therefore suggesting that these tissues may have different functions.

CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor.

Expression of CD200, the gene encoding the ligand for the inhibitory immune receptor CD200R, is an independent prognostic factor for various forms of leukemia predicting worse overall survival of the patients. The enhanced expression of CD200 on the tumors implies that anti-tumor responses can be enhanced by blockage of the CD200-CD200R interaction. Indeed, antibody-mediated blockade of the CD200-CD200R inhibitory axis is currently evaluated in clinical tests to boost immune responses against CD200-expressing tumors. Here, we show that mice lacking CD200, the exclusive ligand for CD200R, are resistant to chemical skin carcinogenesis. Importantly, CD200R controls tumor outgrowth independently of CD200 expression by the tumor cells themselves. Furthermore, Cd200(-/-) mice do not become tolerant to intranasally administered antigens, suggesting that tumor rejection is normally suppressed through CD200-induced immune tolerance. Decreased tumor outgrowth is accompanied by increased expression of the proinflammatory cytokines interleukin (IL)-1ß and IL-6 by the lymph node (LN) dendritic cells. During carcinogenesis, skin-draining LNs of Cd200(-/-) mice contain increased numbers of IL-17-producing FoxP3(+) cells, which preferentially home to the tumors. Thus, the CD200-CD200R axis induces tolerance to external and tumor antigens and influences the T-regulatory/Th17 cell ratio. We demonstrate for the first time that the absence of CD200R signaling inhibits outgrowth of an endogenous tumor irrespective of CD200 expression by the tumor cells. This important paradigm shift leads to a much broader applicability of CD200-blockade in the treatment of tumors.

The interaction of interdigitating cells and lymphocytes in vitro and in vivo.

To investigate the role of the NLDC-145 antigenic determinant on interdigitating cells in T-cell dependent areas of lymphoid organs, rosettes were isolated from interdigitating cells and lymphocytes from lymph nodes. By using allotypic mixtures it was established that these rosettes represented pre-existing associations of interdigitating cells and lymphocytes. Phenotypic analysis of the lymphocytes showed that most cells in these rosettes were T cells. After antigenic stimulation, an increase of helper T cells and of B cells associated with the rosettes was found. Incubation of the rosettes with the NLDC-145 antibody did not alter the composition of the rosettes. By neonatal injection of the NLDC-145 antibody, the influence of the antigen on T-cell differentiation and antigen presenting was investigated. Although the antibody could be detected easily on interdigitating cells and thymus cortex of injected animals, no direct influence of long-term injections of the antibody from birth on antigen presentation and T-cell differentiation was found.

L- and E-selectin can recognize the same naturally occurring ligands on high endothelial venules.

The selectin family of cell adhesion molecules consists of three members, E-selectin (ELAM-1), L-selectin (LECAM-1), and P-selectin (CD62, GMP140, or PADGEM), which are all involved in binding of leukocytes to endothelial cells. All members have structural similarities and they can all bind to a common carbohydrate epitope, sialyl Lewis X in in vitro assays. To study cross-reactivity of the selectins in more detail we used Ig chimeras of murine and human L- and human E-selectin. All three chimeras bound to the natural ligands of L-selectin on specialized high endothelial venules in both human and murine lymphoid organs as determined by immunohistochemistry and were able to precipitate 50- and 90-kDa sulfated ligands from organ cultures of murine peripheral and mesenteric lymph nodes. This recognition was calcium dependent and inhibitable by mAb specific for the lectin domain of the respective selectin. L- and E-selectin binding to high endothelial venules and to the sulfated ligands was inhibitable by the carbohydrates, fucoidan and sialyl Lewis X, respectively. Although immunoprecipitations showed that both murine and human L-selectin could recognize the sulfated ligands from high endothelial venules more efficiently than human E-selectin at the concentrations used, both L- and E-selectin chimeras could inhibit in vivo lymphocyte trafficking into peripheral lymph nodes equally well.

Dendritic cells of the mouse recognized by two monoclonal antibodies.

A new monoclonal antibody, MIDC-8, is described which shows a comparable tissue distribution as the recently described NLDC-145 antibody. It recognizes interdigitating cells, veiled cells and Langerhans cells in lymphoid organs and the skin of the mouse. In contrast to NLDC-145 it recognizes a cytoplasmic component of these cell types. Its distribution is more restricted to the T cell-dependent areas than NLDC-145. Isolation of dendritic cells from lymph nodes, spleen and thymus revealed that both antibodies react with the in vitro isolated dendritic cells. The results show that these antibodies can be used to study dendritic cells in vitro and emphasize the relationship between the in vivo interdigitating cells of the T cell areas and the in vitro isolated dendritic cells.