Lymph Node Subcapsular Sinus Research Articles

Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer.

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Markers of the pre-metastatic niche "knock on the door" of metastasis-free cervical lymph nodes in patients with oral cancer

AimTo assess expression of some markers of the pre-metastatic niche (PMN) in lymph nodes (LNs) of oral cancer patients. MaterialsLNs from metastatic-free neck dissections (LN0/N0, N = 43) and metastatic-free LNs in the vicinity of metastasis-containing LNs (LN0/N+, N = 30) were immuno-histochemically stained for lysyl oxidase (LOX), fibronectin (FN), vascular-endothelial growth factor receptor (VEGFR)-1 and matrix metalloproteinase (MMP)-9. Staining was assessed as 0 (no or weak staining), 1 (strong stain in 25% cells or extracellular area), 2 (same as 1 but in up to 50%) and 3 (same as 1 but in > than 50% of cells/area). Assessment was performed in the lymph node capsule (CAP), sub-capsular sinus (SCS) and medullary sinus (MS). In addition, sections were stained with picrosirius red and examined with polarized microscopy for assessing the distribution of polarization colors of the collagen fibers in the LN capsular area. ResultsAll examined LNs were positive for markers of the PMN. In general, the distribution and intensity of the immunoreactivity was similar between the LN0/N0 and LN0/N+, with only a few differences regarding expression of LOX in the capsule (p = 0.002) and VEGFR1 and MMP9 in the SCS (p = 0.023 and p < 0.001, respectively). Picrosirius red stain and polarized microscopy revealed a disrupted arrangement and distribution of the collagen fibers in both LN0/N0 and LN0/N + . ConclusionMarkers for PMN were shown for the first time to be expressed in cervical LN0/N0 from patients with oral cancer, suggesting the increased permissive pathway remotely paved by the primary oral tumor for the incoming metastatic cells.

Low Molecular Weight Cytokeratin Immunostaining for Extrafollicular Reticulum Cells is an Effective Means of Separating Salivary Gland Tumor-Associated Lymphoid Proliferation from True Lymph Node Involvement

Tumor-associated lymphoid proliferation (TALP) is a well-recognized lymphocytic reaction that is commonly associated with certain salivary gland tumors. A salivary carcinoma with TALP may be confused for true lymph node involvement by that tumor, constituting a potential pitfall in tumor staging that may result in unnecessary therapeutic intervention or erroneous prognostication for patients. True lymph nodes harbor populations of extrafollicular reticulum cells (ERCs), which can be highlighted by low molecular weight cytokeratin immunohistochemistry. We sought to determine whether low molecular weightcytokeratin Cam5.2 immunostaining may be utilized to differentiate true lymph node involvement by salivary gland tumors from TALP. The surgical pathology archives of the University of Texas Southwestern Medical Center was searched forcases of salivary gland neoplasms exhibiting either TALP or true lymph node involvement. Hematoxylin and eosin-stained sections were examined. Cases were classified on the basis of a definitive lymph node capsule and subcapsular sinus, as seen on routine histologic evaluation. Low molecular weight cytokeratin Cam5.2 immunostaining was performed and evaluated on all cases. Twenty-three salivary gland carcinomas with TALP and 16 carcinomas involving a lymph node (14 carcinomas metastatic to regional lymph nodes and 2 carcinomas arising from benign lymph node inclusions) were identified. Numerous Cam5.2-positive ERCs were identified within the nodal tissue of all true lymph nodes involved by carcinoma (16 of 16 cases), while Cam5.2-positive ERCs were completely absent in all cases of salivary gland lesions with TALP (0 of 23 cases) (100% vs. 0%, p < .0001, Fisher's Exact). Utilization of low molecular weight cytokeratin Cam 5.2 immunostaining for ERCs is a highly useful tool for distinguishing true lymph node involvement by salivary gland carcinomas from TALP. This strategy may be useful in identifying genuine nodal metastasis in histologically ambiguous cases, and to avoid erroneously upstaging tumor with TALP as nodal metastasis with the resulting prognostic and therapeutic implications. Moreover, low molecular weight cytokeratin immunostaining may be useful in confirming the rare examples of salivary gland tumors arising from intranodal salivary gland inclusions.

Lymph Node Subcapsular Sinus Macrophages as the Frontline of Lymphatic Immune Defense.

Lymphatic vessels collect and transport lymph and pathogens to the draining lymph node (LN) to generate proper immune protection. A layer of macrophages that strategically line the LN subcapsular sinus (SCS) is directly exposed to the afferent lymph and are denoted as SCS macrophages. These macrophages are the frontline of immune defense that interact with lymph-borne antigens. The importance of these macrophages in limiting the spread of pathogens has been demonstrated in both viral and bacterial infection. In anti-microbial responses, these macrophages can directly or indirectly activate other LN innate immune cells to fight against pathogens, as well as activate T cells or B cells for adaptive immunity. As the first layer of immune cells embracing the tumor-derived antigens, SCS macrophages also actively participate in cancer immune regulation. Recent studies have shown that the LNs' SCS macrophage layer is interrupted in disease models. Despite their importance in fighting the spread of pathogens and in activating anti-tumor immunity, the mechanism and the immunological functional consequences for their disruption are not well-understood. Understanding the mechanism of these macrophages will enhance their capability for therapeutic targeting.

Type I Interferon Signaling to Dendritic Cells Limits Murid Herpesvirus 4 Spread from the Olfactory Epithelium.

Murid herpesvirus 4 (MuHV-4) is a B cell-tropic gammaherpesvirus that can be studied in vivo Despite viral evasion, type I interferons (IFN-I) limit its spread. After MuHV-4 inoculation into footpads, IFN-I protect lymph node subcapsular sinus macrophages (SSM) against productive infection; after peritoneal inoculation, they protect splenic marginal zone macrophages, and they limit MuHV-4 replication in the lungs. While invasive infections can be used to test specific aspects of host colonization, it is also important to understand natural infection. MuHV-4 taken up spontaneously by alert mice enters them via olfactory neurons. We determined how IFN-I act in this context. Blocking IFN-I signaling did not increase neuronal infection but allowed the virus to spread to the adjacent respiratory epithelium. In lymph nodes, a complete IFN-I signaling block increased MuHV-4 lytic infection in SSM and increased the number of dendritic cells (DC) expressing viral green fluorescent protein (GFP) independently of lytic infection. A CD11c+ cell-directed signaling block increased infection of DC only. However, this was sufficient to increase downstream infection, consistent with DC providing the main viral route to B cells. The capacity of IFN-I to limit DC infection indicated that viral IFN-I evasion was only partly effective. Therefore, DC are a possible target for IFN-I-based interventions to reduce host colonization.IMPORTANCE Human gammaherpesviruses infect B cells and cause B cell cancers. Interventions to block virus binding to B cells have not stopped their infection. Therefore, we must identify other control points that are relevant to natural infection. Human infections are difficult to analyze. However, gammaherpesviruses colonize all mammals. A related gammaherpesvirus of mice reaches B cells not directly but via infected dendritic cells. We show that type I interferons, an important general antiviral defense, limit gammaherpesvirus B cell infection by acting on dendritic cells. Therefore, dendritic cell infection is a potential point of interferon-based therapeutic intervention.

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.

Peripheral prion disease pathogenesis is unaltered in the absence of sialoadhesin (Siglec-1/CD169).

Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymphoid tissues, whereas others may aid their clearance by phagocytosis and by destroying them. Our recent data show that an intact splenic marginal zone is important for the efficient delivery of prions into the B-cell follicles where they subsequently replicate upon follicular dendritic cells before infecting the nervous system. Sialoadhesin is an MNP-restricted cell adhesion molecule that binds sialylated glycoproteins. Sialoadhesin is constitutively expressed upon splenic marginal zone metallophilic and lymph node sub-capsular sinus macrophage populations, where it may function to bind sialylated glycoproteins, pathogens and exosomes in the blood and lymph via recognition of terminal sialic acid residues. As the prion glycoprotein is highly sialylated, we tested the hypothesis that sialoadhesin may influence prion disease pathogenesis. We show that after peripheral exposure, prion pathogenesis was unaltered in sialoadhesin-deficient mice; revealing that lymphoid sequestration of prions is not mediated via sialoadhesin. Hence, although an intact marginal zone is important for the efficient uptake and delivery of prions into the B-cell follicles of the spleen, this is not influenced by sialoadhesin expression by the MNP within it.

Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

Lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively by the flow of lymph. We demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node, which requires active tumor cell migration. In human and mouse tissues, CCL1 protein is detected in lymph node lymphatic sinuses but not in the peripheral lymphatics. CCR8, the receptor for CCL1, is strongly expressed by human malignant melanoma. Tumor cell migration to lymphatic endothelial cells (LECs) in vitro is inhibited by blocking CCR8 or CCL1, and recombinant CCL1 promotes migration of CCR8(+) tumor cells. The proinflammatory mediators TNF, IL-1β, and LPS increase CCL1 production by LECs and tumor cell migration to LECs. In a mouse model, blocking CCR8 with the soluble antagonist or knockdown with shRNA significantly decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1-CCR8 in metastasis and lymph node LECs as a critical checkpoint for the entry of metastases into the lymph nodes.

Histopathological atlas and proposed classification for melanocytic lesions in Tyr::NRasQ61K; Cdkn2a−/− transgenic mice

Reference EPFL-ARTICLE-188431doi:10.1111/pcmr.12115View record in Web of Science Record created on 2013-09-09, modified on 2017-05-12

Infection-Induced Regulation of Natural Killer Cells by Macrophages and Collagen at the Lymph Node Subcapsular Sinus

Infection leads to heightened activation of natural killer (NK) cells, a process that likely involves direct cell-to-cell contact, but how this occurs in vivo is poorly understood. We have used two-photon laser-scanning microscopy in conjunction with Toxoplasma gondii mouse infection models to address this question. We found that after infection, NK cells accumulated in the subcapsular region of the lymph node, where they formed low-motility contacts with collagen fibers and CD169(+) macrophages. We provide evidence that interactions with collagen regulate NK cell migration, whereas CD169(+) macrophages increase the activation state of NK cells. Interestingly, a subset of CD169(+) macrophages that coexpress the inflammatory monocyte marker Ly6C had the most potent ability to activate NK cells. Our data reveal pathways through which NK cell migration and function are regulated after infection and identify an important accessory cell population for activation of NK cell responses in lymph nodes.

Abstract 4368: Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

Abstract The lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively, by the flow of lymph. Here, we demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node. In vitro, tumor migration to lymphatic endothelium (LECs) was inhibited by blocking CCR8 or CCL1. Recombinant CCL1 promoted migration of CCR8+ tumor cells. Pro-inflammatory mediators TNF-α, IL-1α and LPS increased CCL1 production by LECs as well as tumor cell migration to LECs. Blocking studies showed that CCL1 is a key molecule mediating tumor cell chemotaxis to inflamed lymphatic endothelium. In mouse and human tissues CCL1 protein was detected in lymph node lymphatic sinuses, but not in the peripheral lymphatics. In addition, CCR8 was strongly expressed by human malignant melanoma. In a mouse model, blocking CCR8 function decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1/CCR8 in metastasis and lymph node LECs as a critical check-point for entry of metastases into the lymph nodes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4368. doi:1538-7445.AM2012-4368

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.

Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.

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).

Visualization of IFNβ production by plasmacytoid versus conventional dendritic cells under specific stimulation conditions in vivo

Type I interferons, a protein family of multiple IFNalphas and a single IFNbeta, initially identified on the basis of their antiviral activities have recently been attributed important roles in bacterial and parasitic infections. To assess the cellular sources of IFNbeta, the IFN produced first in most situations, we created an IFNbeta reporter-knockin mouse, in which yellow fluorescent protein (YFP) is expressed from a bicistronic mRNA linked by an internal ribosomal entry site to the endogenous IFNbeta mRNA. This YFP expression allows spatiotemporal tracking of the initiation of the type I IFN response on a single-cell level. In vitro bone marrow-derived macrophages (BMMPhis) and bone marrow-derived dendritic cells (BMDCs) show IFNbeta production from distinct cell subpopulations in response to defined pathogen compounds. A subpopulation of GMCSF-derived BMDCs produced IFNbeta after poly(I:C), 3'5'-cytidylylguanosine (CpG), or LPS treatment, whereas Flt3-L-cultured plasmacytoid DCs (pDCs) responded mainly to CpG. After poly(I:C) injection in vivo, IFNbeta-producing cells localize to the splenic marginal zone and the lymph node subcapsular sinus. Infection with murine cytomegalovirus (MCMV) induces IFNbeta/YFP expression exclusively in few activated pDCs at the T cell/B cell interface of the splenic white pulp. This IFNbeta/YFP reporter mouse represents a reliable tool for the visualization and characterization of IFNbeta-producing cells in vitro and in vivo.

Antigen Presentation the Macrophage Way

Multiphoton intravital microscopy of lymph nodes has revolutionized the study of the early events leading to induction of acquired immunity. Using this technique, Junt et al. (2007) report in a recent issue of Nature that macrophages of the mouse lymph node subcapsular sinus facilitate B cell activation in vivo by collecting and displaying native antigen.

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.

Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers.

Ligands for the cysteine-rich (CR) domain of the mannose receptor (MR) were detected by incubating murine tissues with a chimeric protein containing CR fused to the Fc region of human IgG1 (CR-Fc). In naive mice, CR-Fc bound to sialoadhesin+, F4/80low/-, macrosialin+ macrophages (M phi) in spleen marginal zone (metallophilic M phi) and lymph node subcapsular sinus. Labeling was also observed in B cell areas of splenic white pulp. Western blotting analysis of spleen and lymph nodes lysates revealed a restricted number of molecules that interacted specifically with CR-Fc. In immunized mice, labeling was upregulated on germinal centers in splenic white pulp and follicular areas of lymph nodes. Kinetic analysis of the pattern of CR-Fc labeling in lymph nodes during a secondary immune response to ovalbumin showed that CR ligand expression migrated towards B cell areas, associated with cells displaying distinctive dendritic morphology, and accumulated in developing germinal centers. These studies suggest that MR+ cells or MR-carbohydrate-containing antigen complexes could be directed towards areas where humoral immune responses take place, through the interaction of the MR CR domain with molecules expressed in specialized macrophage populations and antigen transporting cells.