Marginal Reticular Cells Research Articles

A1.17 A novel role for CD248 in controlling the differentiation of follicular dendritic cells (FDCs) following immune challenge

Background and objectives Follicular dendritic cells (FDCs) are a stromal cell population located within the germinal centre and responsible for antigen presentation and the process of B cell affinity maturation. Although FDC function is well established, the origin and differentiation of these cells is still debated. A recent publication (Jarjour et al . JEM 2014) used multicolour fate mapping techniques to show that lymph node FDC in inflamed lymph nodes derive from marginal reticular cells (MRC), a population of stromal cells that is found underneath the lymph node subcapsular sinus. Contrasting evidence suggests that in the spleen, FDC derive from ubiquitous perivascular precursors (Krautler et al . Cell 2012). The discrepancies in these data could indicate that FDC arise from different precursors, depending on anatomical locations and stimulus. CD248 (also known as tumour endothelial marker-1 or endosialin) is a well-known, pericyte associated, stromal cell marker whose expression is significantly increased in lymph nodes and spleen following immunisation. CD248 is also upregulated in a number of disease states, including in rheumatoid arthritis. In a series of preliminary experiments, we have identified expression of CD248 within the B cell follicles, in the FDC rich area of repetitively immunised cervical lymph nodes. Materials and methods We have immunised CD248 knockout (KO) mice i.p or in the paw pad with the hapten NP-CGG, following an established immunisation protocol that allows tracking of the antigen-specific B cell responses. We analysed the efficiency of the germinal centre response using confocal microscopy, flow cytometry and qPCR, as well as ELISAs to quantify the production of antigen-specific antibodies. Results We present evidence that CD248 plays a role in the formation of FDC networks following immunisation. FDC networks in CD248KO mice lack the normal reticular, dendrite-like structure, despite showing only partial reduction in size. These defective networks associate with a reduction in the ability of CD248KO mice to mount a specific IgG response, implicating CD248 in FDC-mediated control of B cell maturation and production of high affinity antibodies. In vitro CD248KO mice present a defect in mesenchymal stromal cell differentiation toward a lymphoid like phenotype suggesting that CD248 is critical for lymphoid stromal cell formation. Conclusions This data establishes CD248 + pericytes as potential precursors of FDCs. The anatomical location of CD248 + cells within the lymph node; adjacent to the nascent FDCs, identifies CD248 as a candidate for regulation of FDC function in physiological and pathological conditions. Ectopic formation of FDC in chronic autoimmune diseases such as Rheumatoid Arthritis and Sjogren’s syndrome is known to associate with disease progression and worse outcome; targeting CD248 in these conditions could represent a novel and valid therapeutic approach. References Jarjour M, Jorquera A, Mondor I, Wienert S, Narang P, Coles MC, Klauschen F, Bajenoff M. Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells. J Exp Med 2014; 211 (6):1109–22. Krautler NJ, Kana V, Kranich J, Tian Y, Perera D, Lemm D, Schwarz P, Armulik A, Browning JL, Tallquist M, Buch T, Oliveira-Martins JB, Zhu C, Hermann M, Wagner U, Brink R, Heikenwalder M, Aguzzi A. Follicular dendritic cells emerge from ubiquitous perivascular precursors. Cell 2012; 150 (1):194–206.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

Clusterin is a potential lymphotoxin beta receptor target that is upregulated and accumulates in germinal centers of mouse spleen during immune response.

Clusterin is a multifunctional protein that participates in tissue remodeling, apoptosis, lipid transport, complement-mediated cell lysis and serves as an extracellular chaperone. The role of clusterin in cancer and neurodegeneration has been extensively studied, however little is known about its functions in the immune system. Using expression profiling we found that clusterin mRNA is considerably down-regulated in mouse spleen stroma upon knock-out of lymphotoxin β receptor which plays pivotal role in secondary lymphoid organ development, maintenance and function. Using immunohistochemistry and western blot we studied clusterin protein level and distribution in mouse spleen and mesenteric lymph nodes in steady state and upon immunization with sheep red blood cells. We showed that clusterin protein, represented mainly by the secreted heterodimeric form, is present in all stromal compartments of secondary lymphoid organs except for marginal reticular cells. Clusterin protein level rose after immunization and accumulated in light zones of germinal centers in spleen - the effect that was not observed in lymph nodes. Regulation of clusterin expression by the lymphotoxin beta signaling pathway and its protein dynamics during immune response suggest a specific role of this enigmatic protein in the immune system that needs further study.

Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

ILCs in the zone

Innate lymphoid cells, marginal reticular cells and B cell–helper neutrophils interact to promote antibody secretion by B cells in the marginal zone of the spleen in humans and mice.

Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells.

Innate lymphoid cells (ILCs) regulate stromal, epithelial and immune cells, but their impact on B cells remains unclear. We identified RORγt+ ILCs nearby the marginal zone (MZ), a splenic compartment containing innate-like B cells that respond to circulating T cell-independent (TI) antigens. Spenic ILCs established a bidirectional crosstalk with MAdCAM-1+ marginal reticular cells by providing tumor necrosis factor (TNF) and lymphotoxin, and activated MZ B cells via BAFF, CD40 ligand and the Notch ligand, Delta-like 1. Splenic ILCs further helped MZ B cells and their plasma cell progeny by co-opting neutrophils through the release of GM-CSF. Consequently, ILC depletion impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune and circulatory systems.

Nkx2-5+Islet1+ Mesenchymal Precursors Generate Distinct Spleen Stromal Cell Subsets and Participate in Restoring Stromal Network Integrity

Secondary lymphoid organ stromal cells comprise different subsets whose origins remain unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originate from embryonic mesenchymal progenitors of the Nkx2-5(+)Islet1(+) lineage. This lineage include embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable also of supporting ectopic lymphoid-like structures and a subset of resident spleen stromal cells that proliferate and regenerate the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.

Marginal reticular cells: a stromal subset directly descended from the lymphoid tissue organizer

The architecture of secondary lymphoid organs (SLOs) is supported by several non-hematopoietic stromal cells. Currently it is established that two distinct stromal subsets, follicular dendritic cells and fibroblastic reticular cells, play crucial roles in the formation of tissue compartments within SLOs, i.e., the follicle and T zone, respectively. Although stromal cells in the anlagen are essential for SLO development, the relationship between these primordial cells and the subsets in adulthood remains poorly understood. In addition, the roles of stromal cells in the entry of antigens into the compartments through some tissue structures peculiar to SLOs remain unclear. A recently identified stromal subset, marginal reticular cells (MRCs), covers the margin of SLOs that are primarily located in the outer edge of follicles and construct a unique reticulum. MRCs are closely associated with specialized endothelial or epithelial structures for antigen transport. The similarities in marker expression profiles and successive localization during development suggest that MRCs directly descend from organizer stromal cells in the anlagen. Therefore, MRCs are thought to be a crucial stromal component for the organization and function of SLOs.

Role of Nigella sativa in ameliorating chloramphenicol induced tissue damage in rats

Nigella sativa ascribed to have many medicinal properties. The study aimed to investigate whether N. sativa oil could decrease the side effects induced by the antibiotic, chloramphenicol. Rats were assigned into the following: The control group (first group); the second and the third groups which were orally administrated chloramphenicol for 21 days at a dose of 86 mg/kg body weight. Then the third group was treated with N. sativa oil for 30 days at a dose of 13.5 mg/ 150 g; the forth group was synergistically administrated both chloramphenicol and N. sativa for 21 days. Administration of N. sativa oil was extended to 30 days more in this group. There was a decrease in erythrocyte, hemoglobin and hematocrit with a progressive increase of leukocyte count in drug treated group. A decrease in neutrophils and lymphocyte with an increase in nucleated immature red cells as well as myeloblasts and myelocytes. On the other hand, N. sativa showed a time dependent improvement in blood parameters. A gradual decrease in the counts of immature stages was realized with the administration of N. sativa. Pathological changes in spleen included splenomegaly, lymphocytic depletion, enlargement of the marginal zone, wide trabeculae, reticular cells, pyknotic nuclei and cells in different stages of megakaryopoiesis. A marked depletion in cortical lymphocytes, disturbed lobular pattern, increased reticuloepithelial cells and dilated blood vessels in the thymus of group 2. N. sativa showed only a slight improvement of the damaged spleen tissues, while a time dependent repair in thymic tissue and both the cellular and humoral immunity was observed. In conclusion, N. sativa had an obvious protective effect and decreased the side effects of chloramphenicol. Key words: Nigella sativa, chloramphenicol, pathological changes.

Reproducible Isolation of Lymph Node Stromal Cells Reveals Site-Dependent Differences in Fibroblastic Reticular Cells

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Migration of immature and mature B cells in the aged microenvironment

Studies in aged mice show that the architecture of B-cell areas appears disrupted and that newly made B cells fail to incorporate into the spleen. These observations may reflect altered migration of immature and mature B cells. Using adoptive transfer, we tested the effect of the aged microenvironment and the intrinsic ability of donor B cells from aged mice to migrate to spleens of intact hosts. Spleens of aged recipients were deficient in attracting young or old donor immature B cells. In contrast, immature and mature B cells maintained an intrinsic ability to migrate to young recipient spleens, except that as the aged immature B cells matured, fewer appeared to enter the recirculating pool. CXCL13 protein, which is necessary for the organization of B-cell compartments, was elevated with age and differences in CXCL13 distribution were apparent. In aged spleens, CXCL13 appeared less reticular, concentrated in patches throughout the follicles, and notably reduced in the MAdCAM-1(+) marginal reticular cells located at the follicular edge. Despite these differences, the migration of young donor follicular B cells into the spleens of old mice was not impacted; whereas, migration of young donor marginal zone B cells was reduced in aged recipients. Finally, the aged bone marrow microenvironment attracted more donor mature B cells than did the young marrow. Message for CXCL13 was not elevated in the marrow of aged mice. These results suggest that the aged splenic microenvironment affects the migration of immature B cells more than mature follicular B cells.

Organizer-Like Reticular Stromal Cell Layer Common to Adult Secondary Lymphoid Organs

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.

A common system of sparsely-branched projection (reticular) NADPH-diaphorase neurons in formations of densely-branched cells in the human forebrain

Morphometric studies of human forebrain formations composed of densely branched cells - the entorhinal cortex, the basolateral amygdala, the nucleus accumbens, the striatum, and the dorsal thalamus - were performed using nine parameters, with statistical analysis of the resulting data; measurements addressed the major projection-type densely branched and sparsely branched reticular neurons (scattered reticular and marginal reticular cells of the dorsal thalamus) stained by the Golgi method and with NADPH-diaphorase. Scattered reticular cells in the various formations showed no differences in any of the nine measures, while there were significant differences (in 5-7 measures, apart from one comparison, where there were differences in two measures) in their major projection-type densely branched cells. Scattered reticular and main projection-type densely branched neurons in each formation differed in terms of 7-9 measures. In endbrain formations, scattered reticular neurons contained NADPH-diaphorase; in the dorsal thalamus, only intermediate marginal reticular neurons were NADPH-diaphorase-positive. Thus, these human formations contained a common system of ancient integrative NADPH-diaphorase-containing reticular cells. Our results, along with published data, show these to be projection-type cells with projections to layers V and VI of the neocortex, which suggests that they have modulatory influences on its descending systems.