Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

Development of antigenic heterogeneity in the splenic meshwork of severe combined immunodeficient (SCID) mice after reconstitution with T and B lymphocytes

Recently, we produced monoclonal antibodies reacting specifically with the reticular meshwork (RM) of lymphoid tissues, and demonstrated that, in the splenic white pulp of normal mouse, the antigenic heterogeneity of RM was associated with the segregation of the T and B lymphocytes. In the present study, we attempted to visualize further the interaction between splenic RM and T and B lymphocytes transferred into severe combined immunodeficient (SCID) mice. The splenic white pulp of naive SCID mice, containing a few T and B cells, showed little tendency for T-B segregation and antigenic diversity of RM. Transfer of spleen or bone marrow cells from normal mice resulted in complete recovery of lymphocyte populations, showing not only a clear segregation of T and B lymphocytes but also a remarkable antigenic diversity of RM. The same results were obtained following the transfer of spleen or bone marrow cells from the nude mouse. Next, we transferred purified T lymphocytes to one group of SCID mice and B cells to another. In mice given T cells, a few B cells were observed in the white puop; T lymphocytes lodged not only in the inner periarterial lymphatic sheath (PALS) but also in the outer PALS and follicles. In the animals to which B cells were transferred, T cells were few and the homing of B cells occurred only into their proper compartments, such as the outer PALS, follicles and marginal zone, but not in the inner PALS. Thus, B cells can home into their proper compartments of the splenic white pulp independently of T lymphocytes.     

Scanning- and transmission electron-microscopic study of lymphatic vessels in the splenic white pulp of the macaque monkey

Summary The fine structure of the lymphatic vessels in splenic white pulp of the macaque monkey was studied by scanning and transmission electron microscopy.Lymphatic vessels were slit-like or widened channels which extended along central arteries and their large branches. The walls of the vessels were very thin in comparison with those of nearby arteries. They were composed only of a layer of endothelium supported by underlying reticular cells. Endothelial cells were mostly ribbon-like and extended along the long axis of the vessels. Perikarya of the endothelial cells were slightly protruded into the lumen. The thin peripheral cytoplasm showed smooth surfaces, except for some tiny processes, especially at boundaries between adjacent cells. The basal surface of the endothelial cells was attached to the lattice of reticular cell processes forming the framework of the white pulp. Basal laminae in strands were intercalated between endothelial cells and reticular cells. Perforations were often seen through the endothelial cell cytoplasm. Lymphocytes or processes of macrophages seen in the perforations were considered to be in migration. Large patent openings through the endothelium were not observed. The wall structure of the lymphatic vessels in the splenic white pulp suggests that lymphocytes in the white pulp may move directly into the lymph flow, in addition to moving into the blood flow via the vascular sinuses.Supported by Research Grant-in Aid from the Ministry of Education, Japan (Grant NO. 56480081).     

Local lymphogenic migration pathway in normal mouse spleen

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP⁺) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP⁺ cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.     

Thymic and splenic changes following injection of living Brucella and BCG in the Guinea pig

Summary The thymic and splenic reactions, following injections of BCG and living Brucella M. in the guinea pigs, were studied. Both microorganisms injected intravenously produced thymic involution, maximal after BCG inoculation, followed by regeneration that was complete by day 10 in Brucella M.-treated guinea pigs, and by day 15, in BCG injected guinea pigs. Increase of mitotic index was more accentuated and more persistent after Brucella M. than after BCG treatment in the thymus. After a short involution period the spleen of injected animals increased in size in both groups. The splenic enlargement was dramatic and occurred at an accelerated rate in animals given BCG. It appeared to be the result of a conspicuous involvement of the red pulp by multiple granulomas. In Brucella M. treated guinea pigs the splenic enlargement was less obvious, but the splenic white pulp was more abundant in BCG-treated guinea pigs. Granulomas were observed only in the periarterial sheaths of the white pulp.These observations provide evidence for the hypothesis that injections of both BCG and Brucella M. provoke a proliferation of B and T lymphocytes, a migration of T lymphocytes from the thymus to the T-dependent area of the spleen which seems, perhaps, more marked after injection of Brucella M., and a strong granulomatous histiocytic reaction which is more conspicuous in animals given BCG.     

Rapid induction of splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2 antigen

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.     

The three-dimensional structure of human splenic white pulp compartments.

The precise arrangement of B- and T-lymphocytes in the different compartments of the human splenic white pulp is still largely unknown. We therefore performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The results indicate that the T-cell regions of human spleens may be interrupted by B-cell follicles. Therefore, there is no continuous periarteriolar lymphatic T-cell sheath (PALS) around white pulp arterioles. An arteriole may be surrounded by T-lymphocytes at one level, then run across a follicle without any T-cells around, and finally re-enter a T-cell region. T- and B-cell compartments are intricately interdigitated in the human splenic white pulp. CD4(+) T-lymphocytes and the typical fibroblasts of the T-cell region may extend as a thin shell at the follicular surface within the marginal zone. On the other hand, IgD(++) B-cells continue from the follicular outer marginal zone along the surface of the T-cell region. Our findings indicate that the microanatomy of the splenic white pulp differs between humans and rodents. This may have consequences for the immigration of recirculating lymphocytes and for initial interactions among antigen-specific T- and B-lymphocytes.     

Electron-microscopic study of the development of the periarteriolar zone in splenic white pulp of rats

Summary To obtain more information concerning the origin of interdigitating cells, the postnatal development and morphology of the periarteriolar lymphatic sheath in splenic white pulp of rats was investigated by light- and electron-microscopy. Special attention was paid to the ontogeny of interdigitating cells. The spleens of the animals were studied in the age range from 1 h to 28 days after birth.The splenic white pulp of neonatal rats consists only of a few reticuloblasts, which are concentrically arranged around central arterioles. After 21 h an increase in promonocytes and monocytes was noted. Between the fifth and seventh postnatal day monocytogenic cells with a light and almost translucent cytoplasm appear, which display long cytoplasmic projections between the adjacent cells. Neighbouring lymphocytes often insert finger-like processes into the invaginated cellular membrane of these transitional forms. This intimate cellular contact is supported by zonulae occludentes. These cells represent transitional forms between monocytes and interdigitating cells.From seven days of age onwards typical interdigitating cells were present as in adult animals. After the differentiation into an inner and outer periarteriolar lymphatic sheath, the T-cell-dependent area of splenic white pulp has attained its adult appearance and further changes are not to be expected.On the basis of these findings, it is highly probable that interdigitating cells develop via transformation of monocytes.This investigation was supported by the Deutsche Forschungsgemeinschaft (^*He 537)     

鳗鲡肝脏、脾脏显微与超微结构

经光镜和电镜观察发现：鳗鲡肝脏的肝小叶不规则。肝细胞胞质内富含多种细胞器及包含物。胆小管由２—４个肝细胞围成，相邻肝细胞间有连接复合体封闭胆小管。肝血窦为有孔型、孔处无隔膜，内有巨噬细胞。窦周隙明显，未见贮脂细胞。肝细胞向胆小管腔与窦周隙面伸出许多指状微绒毛。脾脏内白髓中淋巴细胞聚集成群，未见明显脾小结、淋巴鞘。红髓由脾索与脾窦组成，动脉分支末端（壁厚的毛细血管）可开放于红髓，无明显巨噬细胞中心。脾窦及脾小动脉内皮细胞通常为长杆状、沿血管纵向平行排列。脾窦为有孔型，孔处可见薄的隔膜。脾小动脉内皮外为２—５层平滑肌（多数为纵行）。     

The cellular response of T and B dependent areas of the spleen after focal thermocoagulation of liver and kidney

Using 3H-thymidine autoradiography, the cellular response of the white splenic pulp after single or repeated thermolesions on liver and kidney was studied. Sham operations served as controls, and did not induce any significant changes in the splenic white pulp. After single or repeated thermolesions, the labeling index increased distinctly in the B and T cell areas. The central T-dependent periarteriolar lymphatic sheath (PALS) showed an earlier rise of radioactively labeled lymphocytes after repeated thermolesions than after a single lesion. After a single heat coagulation of liver and kidney a dissociation of the germinal centers occurred with consecutive hyperplasia. However, compared with the splenic reaction after other surgical injuries, especially after cryosurgical operations, the response of the B-dependent splenic areas was weaker. The distinct reaction of the central T-dependent PALS supports the conception of a more prominent cell mediated immune response of the peripheral lymphatic system with temporary T cell dysfunctions. Possibly the inflammatory complications of burned patients and the delayed wound healing of thermosurgical tissue lesions may be caused by the alteration of the lymphatic system with a disturbed cooperation of T and B lymphocytes with macrophages.     

Plasmodium chabaudi chabaudi infection in mice induces strong B cell responses and striking but temporary changes in splenic cell distribution

B cells and Abs play a key role in controlling the erythrocytic stage of malaria. However, little is known about the way the humoral response develops during infection. We show that Plasmodium chabaudi chabaudi causes major, but temporary changes in the distribution of leukocytes in the spleen. Despite these changes, an ordered response to infection develops, which includes vigorous extrafollicular growth of plasmablasts and germinal center formation. Early in the response, the lymphocytes in the T zone and follicles become widely spaced, and the edges of these compartments blur. This effect is maximal around the peak of parasitemia. Germinal centers are apparent by day 8, peak at day 20, and persist through day 60. Extrafollicular foci of plasmablasts are visible from day 4 and initiate a very strong plasma cell response. Initially, the plasma cells have a conventional red pulp distribution, but by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. In this region they form clusters occupying part of the area normally filled by T cells. B cells are absent from the marginal zone for at least 30 days after the peak of infection, although flow cytometry shows their continued presence in the spleen throughout infection. Relatively normal splenic architecture is regained by day 60 of infection. These results show that the changes in splenic cell distribution are linked to the presence of parasites and do not seem to interfere with the development of the humoral response.     

Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen

Summary Male Wistar rats were injected intravenously with 5-(³H)uridine-labeled lymphocytes isolated from lymph nodes of syngeneic donors and enriched in T cells. After short periods of time (3 to 120 min after injection), labeled lymphocytes were localized in spleen compartments using autoradiography to identify routes of lymphocyte movement from blood into splenic parenchyma and to follow migration pathways of recirculating lymphocytes within the periarterial lymphoid sheath (PALS). Topographical analysis of labeled lymphocytes was performed in specific planes of PALS characterized by the diameter of the arterial vessel and termed PALS large, PALS medium, and PALS small (PALS L, PALS M, PALS S, respectively). Attention was also paid to accumulations of labeled lymphocytes close to the arterial vessel wall. Initially, labeled lymphocytes were localized in PALS S and PALS M near the terminal branching of arterial vessels and in the marginal zone (MZ). We conclude that lymphocytes emigrate from blood into splenic parenchyma within two white pulp compartments: in MZ, and directly within PALS through the wall of capillary vessels. The sequential accumulation of labeled cells near arterial vessels of increasing diameter suggests that the recirculating pool of lymphocytes migrates into the central part of PALS L by two routes: from MZ, and along arterial vessels from PALS S and PALS M.R.B. was a fellow of the Alexander von Humboldt-Stiftung, on leave from the Department of Histology and Embryology, Institut of Biostructure, Academy of Medicine, ul. Swiecickiego 6, PL-60-781 Pozna, Poland.     

Immune proteasomes in the developing rat spleen

Changes in the structure of the rat spleen and the distribution of immune proteasomes in it during early postnatal development have been studied using double immunofluorescent staining of tissue sections with antibodies to the LMP7 immune proteasome subunit and to specific markers of T and B lymphocytes. It has been shown that the white pulp on postnatal day 5 is not yet colonized by lymphocytes and contains a smaller amount of immune proteasomes than the red pulp. At this stage, T and B lymphocytes concentrate mainly in the red pulp. On day 8, B lymphocytes occupy the marginal zone, while T lymphocytes aggregate into dense strands close to the white pulp. By day 18, T lymphocytes form periarteriolar sheaths in the white pulp, and the contents of immune proteasomes in the red and white pulp become equally high. An increase in the total content of immune proteasomes in the spleen on the third postnatal week was revealed in our previous study by Western blotting. In addition to T and B lymphocytes, immune proteasomes have also been revealed in other spleen cell types, probably in macrophages and reticular cells of the white pulp. Thus, the postnatal development of the spleen is associated with an increase in the contents of immune proteasomes in it.     

Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.     

Lymphocyte traffic through lymph nodes and Peyer's patches of the rat: B- and T-cell-specific migration patterns within the tissue,and their dependence on splenic tissue

The migration routes of lymphocyte subsets through organ compartments are of importance when trying to understand the local events taking place during immune responses. We have therefore studied the traffic of B, T, CD4⁺, and CD8⁺ lymphocytes through lymph nodes and Peyer's patches. At various time points after injection into the rat, labeled lymphocytes were localized, and their phenotype characterized in cryostat sections using immunohistochemistry. Morphometry was also performed, and the recovery of ⁵¹Cr-labeled lymphocytes in these organs was determined. B and T lymphocytes entered the lymph nodes via the high endothelial venules in similar numbers. Most B lymphocytes migrated via the paracortex (T cell area) into the cortex (B cell area), and then back in substantial numbers into the paracortex. In contrast, T lymphocytes predominantly migrated into the paracortex and were rarely seen in the cortex. No obvious differences were seen between various lymph nodes and Peyer's patches and the routes of CD4⁺ and CD8⁺ lymphocytes. After injection of lymphocytes into animals with autotransplanted splenic tissue, the number of B lymphocytes that had migrated into the B cell area of lymph nodes and of Peyer's patches was significantly decreased, whereas CD4⁺ lymphocytes migrated in larger numbers into the T cell area of both organs.This study was supported by the Deutsche Forschungsgemein-schaft (SFB 244, A7).     

Constitutive expression of CCR7 directs effector CD8 T cells into the splenic white pulp and impairs functional activity

Antigenic stimulation down-regulates CCR7 on effector T cells. To analyze the importance of CCR7 down-regulation, transgenic (tg) mice constitutively expressing CCR7 were generated. CD8 T cells with defined Ag specificity were obtained by breeding CCR7-tg mice with P14 TCR-tg mice specific for lymphocytic choriomeningitis virus. Transgenic CCR7 expression did not impair proliferation of P14.CCR7 T cells induced by lymphocytic choriomeningitis virus infection, but prevented CCR7 down-regulation. Compared with wild-type P14 effector cells, P14.CCR7 effector cells, expressing the CCR7 transgene, were increased in the spleen, but decreased in blood and peripheral tissues. Moreover, P14.CCR7 effector cells localized almost exclusively in the splenic white pulp, whereas P14 effector cells were excluded from splenic white pulp cords and were found preferentially in the red pulp. Functional experiments further revealed that P14.CCR7 effector cells were impaired in rapid viral clearance and in inducing Ag-specific delayed-type hypersensitivity reactions. Thus, the present study demonstrates that down-regulation of CCR7 during CD8 T cell activation is important to release effector cells from the white pulp of the spleen, and highlights the importance of effector cell localization in providing rapid immunity.     

Ultrastructure of splenic white pulp of the turtle,Mauremys caspica

Summary The ultrastructure of splenic tissue of non-immunized turtles, Mauremys caspica, shows two areas, namely, the white pulp which is lymphoid in nature, and the red pulp which is formed by cell cords and sinusoids. Between both areas there is always a marginal zone with gaps through which cells leak. In the white pulp, there are two blood vessel types; one with muscled walls, and the other showing thinner walls sheathed by reticular cells. Reticular cells constitute a network where there occur dendritic macrophages, lymphoblasts and small and medium lymphocytes. Mature plasma cells are scarce in the white pulp.     

PrP-dependent association of prions with splenic but not circulating lymphocytes of scrapie-infected mice.

An intact immune system, and particularly the presence of mature B lymphocytes, is crucial for mouse scrapie pathogenesis in the brain after peripheral exposure. Prions are accumulated in the lymphoreticular system (LRS), but the identity of the cells containing infectivity and their role in neuroinvasion have not been determined. We show here that although prion infectivity in the spleen is associated with B and T lymphocytes and to a lesser degree with the stroma, no infectivity could be detected in lymphocytes from blood. In wild-type mice, which had been irradiated and reconstituted with PrP-deficient lymphohaematopoietic stem cells and inoculated with scrapie prions, infectivity in the spleen was present in the stroma but not in lymphocytes. Therefore, splenic B and T lymphocytes can either synthesize prions or acquire them from another source, but only when they express PrP.     

The anatomy of peripheral lymphoid organs with emphasis on accessory cells: light-microscopic immunocytochemical studies of mouse spleen, lymph node, and Peyer's patch

Antigen, lymphocytes, and accessory cells interact within peripheral lymphoid organs to generate immunity. Two cell types have been studied for accessory function in culture: mononuclear phagocytes and nonphagocytic Ia-rich dendritic cells. The monoclonal antibodies which have been used to study isolated murine macrophages (M phi) and dendritic cells (DC) include alpha-macrophage (F4/80, M1/70), alpha-dendritic cell (33D1), alpha-Fc receptor (2.4G2), and alpha-Ia (B21-2) reagents. In this paper, the antibodies have been used to stain accessory cells in cryostat sections of mouse spleen, lymph node, and Peyer's patch. Each organ is known to contain subregions that are rich in either macrophages, B cells, or T cells. We found that the accessory cells in each subregion had a different phenotype. 1) Macrophage-rich regions: Macrophages that lined the site of antigen delivery (marginal zone of spleen, around afferent lymphatics of node, and below the epithelium of Peyer's patch) were stained with M1/70 but not with F4/80. F4/80 was abundant on macrophages in other sites: spleen red pulp, node medulla, and around Peyer's patch efferent lymphatics. 2) B-lymphocyte-rich follicles: Follicular dendritic cells, which retain immune complexes extracellularly, are concentrated on the outer aspect of the germinal center. This region stained strongly with alpha-Fc receptor antibody 2.4G2, but not with M1/70, F4/80, or 33D1. 3) T areas: The interdigitating cells of T areas have been linked to isolated dendritic cells. Irregular Ia-rich cells were distributed uniformly in the T areas of each organ. However, staining with 33D1 was not detected and was restricted to foci of nonphagocytic cells at the spleen red/white pulp junction. F4/80, M1/70 or 2.4G2 also did not stain the T area, except for the region close to splenic central arteries. Therefore the principal surface markers and locations of the candidate accessory cells in murine lymphoid organs are M1/70+ macrophages at the site of antigen entry; F4/80+ macrophages around regions of lymphocyte efflux; germinal center dendritic cells, which may be rich in 2.4G2; and Ia-rich interdigiting cells in the T area.     

Intra-thymic/splenic engraftment of human T cells in HLA-DR1 transgenic NOD/scid mice

A HLA-DR1 transgenic mouse (NOD/scid-DR1) was derived by breeding the existing B10.M/J-[Tg]DR1 mouse with the NOD/scid mouse. The intention was to enhance engraftment of human T cells by providing human class II elements in the tissues. Thymus and spleen fragments from adult NOD/scid-DR1 mice were transplanted under the syngeneic kidney capsules, followed by injection of human cord blood mononuclear cells (CBMNC) into transplanted tissues. FACS analyses showed that human T and B cells were consistently detected in the peripheral blood and spleen, of the chimeric mice. An average of 20% of human cells was found in the spleen and the engrafted thymus/spleen tissues. Furthermore, human cells from these tissues could proliferate with anti-human CD3 antibody and these mice could generate humoral and cellular responses to allogeneic human cells. Cytokines, such as IL-10, GMCSF, IFN-gamma, and TNF-alpha were also detected in the supernatants of the cultured human cells from the chimeric mice, when they were stimulated with allogeneic cells. Therefore, a novel mouse model with functional circulating human T and B cells was established that would facilitate the exploration of vaccine, the disease processes of autoimmunity, HIV infection, and human cancer.