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Datasets  >  imaging dataset  >  Studying the cellular organ...

Studying the cellular organization of the marrow of long bones by Immunohistochemistry and Confocal Microscopy.

imaging dataset

DC-THERA applicant : Anita Sapoznikov
Institution: The Weizmann Institute of Science, Israel
Supervisor: Dr. Steffen Jung

Data has been obtained on the cellular organization of the marrow of long bones.

The bone marrow (BM) is a primary lymphoid organ, where diverse hematopoietic lineages arise from common multi-potential progenitors [1]. However, aside from this established role in hematopoiesis, the BM also serves as secondary lymphoid organ in the generation of adaptive T and B cell immune responses [2,3]. It has been shown that the BM is also populated by mature lymphocytes, which home to the BM and seem to inhabit specific niches [2,4]. Two photon microscopic analysis of mouse skull BM revealed that the BM-resident Dendritic cells (bmDC) in the cranial BM parenchyma are organized in unique peri-vascular clusters tightly wrapped around distinct BM blood vessels and occupying architecturally definable niches. Interestingly, these clusters are reminiscent of recently reported peri-sinusoidal niches that are defined by mature re-circulating B cells [2]. We showed that grafted B and T cells efficiently homed to the BM and their distribution was restricted to the extra-vascular areas covered by bmDC. Using a conditional in vivo DC ablation system, we analyzed the BM and periphery of DC-depleted mice and found that bmDC ablation led to a profound B cell reduction. The effect was specific to the BM, since the frequencies of B cells in the spleen and lymph nodes were not impaired. Moreover, the reduction was specific for recirculating mature B cell compartment, whereas B cell development was unaffected by bmDC ablation. This phenotype could be reverted by B cell over-expression of the anti-apoptotic factor bcl2. Moreover, we showed that re-circulating B cell survival requires the presence of bmDC, which produce macrophage migration inhibitory factor (MIF) in the perivascular BM niches [5].

The aim of my visit at the Deutsches Rheuma-Forschungszentrum (DRFZ) and the laboratory of Prof. Andreas Radbruch was to study the organization of bmDC, B lymphocytes and plasma cells in the BM of the long bones by immunochistochemistry. During my visit I learned the preparation of the bones for histology, crysectioning, immunohistochemistry and analysis of the stained sections by confocal microscope.
Using a CX3CR1GFP mice, in which bmDC are green fluorescent (GFP), I analyzed the bmDC localization and found the the cells organized in unique clusters, mainly located in the endosteum of the BM. These DC co-localize with B cells and these clusters are occupying architecturally definable niches and are reminiscent to the niches that were found in the cranial BM parenchyma. By using Fibronectin antibody I could visualize the stroma of the BM. In addition, a large fraction of IgA positive plasma cells is located in the BM. Staining for these cells revealed that plasma cells are scattered through the BM and are not preferentially found in DC-B cell niche. However, depletion of DC led to reduction in IgA+ plasma cells, which might indicate that bmDC are critical for survival of these cells in the BM. Thus, additional experiments will be conducted in order to understand the interactions between bmDC and plasma cells.

1. Nagasawa, T. et al. Microenvironmental niches in the bone marrow required for B-cell development. Nature Immunol. 6, 107-116 (2006)
2. Cariappa, A. et al. Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes. Immunity 23, 397-407 (2005)
3. Cavanagh, L. et al. Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nature Immunol. 6, 1029-1037 (2006)
4. Tokoyoda, K. et al. Cellular niches controlling B lymphocytes behavior within bone marrow during development. Immunity 20, 707-718 (2004)
5. Sapoznikov A. et al. Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches. Nat. Immunol. 9, 388-395 (2008)

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