Are murine marginal-zone macrophages the splenic white pulp analog of high endothelial venules?

The entry of lymphocytes into the spleen, in contrast to lymph nodes, does not involve high endothelial venule (HEV) interaction. The precise point of entry, as well as the mechanism by which lymphocytes enter the lymphoid areas of the spleen, remains controversial. We examined in detail the effect of two agents, pertussis toxin (PT) and the sulfated polysaccharide fucoidan, on splenic lymphocyte entry and positioning. These have previously been shown to interfere with lymphocyte extravasation across HEV. PT prevents lymphocyte extravasation, but not binding, to HEV, whereas fucoidan prevents binding and thus subsequent extravasation. Studies presented here show that pretreatment of murine lymphocytes with PT does not numerically affect entry into spleen, but profoundly alters lymphocyte positioning within the spleen. When fluorescently labeled, PT-treated lymphocytes are injected intravenously, they initially accumulate in the marginal zone, in apparent association with the layer of marginal zone macrophages (MZM?) which form a shell around the white pulp. They fail to traverse this layer into the white pulp, and subsequently localize in the red pulp. In contrast, untreated cells initially appear in the marginal zone, then continue to migrate into the white pulp after traversing the MZM? layer. The localization of PT-pretreated lymphocytes adjacent to the MZM? layer is disrupted by intravenous administration of fucoidan. Using a flow cytometric assay of aggregation between MZM? and lymphocytes, we confirmed that fucoidan is also able to inhibit this association in vitro, whereas PT has no effect on this interaction. We propose that MZM? in the mouse are the splenic analog of HEV, forming the port of entry of lymphocytes into the white pulp of the spleen.     

Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp, Litopenaeus vannamei

Although numerous mechanisms of immune defense have been described in crustaceans, the tissue distribution and fate of live bacteria introduced into the host remain unclear. In the present study, Litopenaeus vannamei were injected with a sub-lethal dose of kanamycin-resistant Vibrio campbellii expressing green fluorescent protein. Accumulation of intact bacteria was quantified by real-time PCR, while bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. Over the 240 min examined, the lymphoid organ contained the greatest number of intact V. campbellii per gram tissue as well as the lowest percentage of culturable V. campbellii compared to other tissues, including the hemolymph. In contrast, the gills and hepatopancreas accumulated intact bacteria, but contained a significantly greater percentage of culturable bacteria than the hemolymph after 240 min. These data suggest that the lymphoid organ plays a major role in bacterial uptake and bacteriostasis in penaeid shrimp.     

The rat FcR for monomeric IgG is preferentially expressed on red pulp macrophages in the spleen.

Rat monoclonal IgG2b immunoglobulins labelled with 125I or biotin were localized in the red pulp areas but were not found in the marginal zones of the white pulp of the spleen 8 hr after their i.v. injection. In cell suspensions made from the spleen, 90% of red pulp macrophages (M phi) bound 125I-IgG2b monomeric proteins in vitro, whereas similar estimates for marginal zone M phi were 12-30%. Red pulp and marginal zone M phi were distinguished primarily by monoclonal antibodies (mAb) ED2 and ED3, but also by their ability to take up FITC-labelled Ficoll in vivo.     

Separation of splenic red and white pulp occurs before birth in a LTalphabeta-independent manner

For the formation of lymph nodes and Peyer's patches, lymphoid tissue inducer (LTi) cells are crucial in triggering stromal cells to recruit and retain hematopoietic cells. Although LTi cells have been observed in fetal spleen, not much is known about fetal spleen development and the role of LTi cells in this process. Here, we show that LTi cells collect in a periarteriolar manner in fetal spleen at the periphery of the white pulp anlagen. Expression of the homeostatic chemokines can be detected in stromal and endothelial cells, suggesting that LTi cells are attracted by these chemokines. As lymphotoxin (LT)alpha1beta2 can be detected on B cells but not LTi cells in neonatal spleen, starting at 4 days after birth, the earliest formation of the white pulp in fetal spleen occurs in a LTalpha1beta2-independent manner. The postnatal development of the splenic white pulp, involving the influx of T cells, depends on LTalpha1beta2 expressed by B cells.     

The role of lymphoid tissue inducer cells in splenic white pulp development

CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are crucial for the development and organisation of lymph nodes and gut associated lymphoid tissues. In this report, we characterise their appearance in the developing spleen and highlight their importance in relation to the development of splenic T cell zones. LTi cells were detected in embryonic spleen from embryonic day 13, although their progenitors were present at embryonic day 12. These cells clustered initially around splenic blood vessels in a lymphotoxin (LT)-independent manner, but up-regulation of VCAM-1 expression on stromal cells associated with the blood vessels was LT dependent. After birth, T cell colonisation of these clusters to form nascent white pulp areas was also LT dependent. Transfer experiments reconstituting RAG(-/-) mice with either WT or LTalpha(-/-) splenocytes demonstrated that lymphocyte expression of LT was not essential for the organisation of a discrete CD3(+) T cell zone with localised podoplanin and CCL21 expression. Our studies indicate that a combination of LT signals from LTi cells and LT-independent signals from lymphocytes is sufficient for expression of podoplanin and CCL21 on splenic T cell zone stroma and subsequent T cell organisation.     

The white pulp in the setting of the septic spleen caused by different bacteria: a comparative morphometric study

In the past little attention has been paid to histomorphologic changes accompanying the phenomenon of the septic spleen, thus indirectly reinforcing the old axiom that the spleen is an organ of mystery. It is especially noteworthy that the relationship between different causative bacteria and histopathologic abnormalities of the white pulp has not been investigated. In this study morphometric analysis was performed on the white pulp of 30 spleens obtained at autopsy from individuals with premortal sepsis. A strictly defined age- and sex-matched control group was analyzed for statistical comparison. Our findings demonstrate a significant depletion of B- and T-areas in the septic spleen, accompanied by a significant tendency towards reactive germinal center hyperplasia regardless of the type of bacteria responsible. However, depletion of splenic B-areas was shown to be significantly pronounced in the setting of premortal enterococcemia in comparison with a panel of gram-negative flagellated bacteria. It is felt that certain bacterial virulence factors (e.g. flagellation and/or structural components of the cell wall) might be pathogenetically involved in the observed changes, reflecting a partially different activation of splenic lymphocytes in the setting of the septic spleen.     

Endotoxin-induced appearance of peroxidase-positive cells in the white pulp of the mouse spleen.

Endotoxin (1-10 microgram intravenously) increased the number of strongly peroxidase-positive cells in the white pulp of the mouse spleen, demonstrable by the Graham-Karnovsky technique. The increase was greatest after 10 h, the cells being found most frequently around the central vessels of the white pulp. The appearance of such cells at this critical immunological site may relate to the known adjuvant activity of endotoxin in promoting immune responses against protein antigens.     

The amount of white pulp in the spleen; a morphometrical study done in methacrylate-embedded splenectomy specimens

This report deals with a morphometrical study on 92 surgically removed human spleens, to investigate the composition of spleens which are considered to be normal, i.e. spleens which had ruptured in traffic accidents or which had been incidentally removed during abdominal operations. A comparison was made with 16 spleens with hypersequestration of platelets and to 11 with hyper-sequestration of erythrocytes. Methyl-methacrylate embedding was used because of the superiority of this technique over conventional paraffin embedding. Significant differences were found between both 'normal' groups as to the absolute and relative amount of white pulp as well as the perifollicular red pulp zone. Based also on the few morphometrical reported studies in the literature, spleens removed during abdominal surgery form the best control group. Traumatic rupture of the spleen in traffic accidents might specifically occur in spleens which already contained a stimulated lymphatic compartment. A probably non-specific increase of white pulp was found in splenomegaly of varied aetiology. An expected influence of age on weight and composition of the spleen was not found in our study. The spleen changes in weight and composition only up to 5 years of age. Significant involution at older age was not found in ours nor in other reported larger series.     

An antibody reacting with splenic red pulp macrophages in the sera of patients with rheumatic diseases

An antibody was detected in the sera of patients with certain rheumatic diseases that reacted with the cytoplasm of the splenic red pulp (SRP) cells of adult mice. This antibody was detected in the sera of all patients with mixed connective tissue disease (MCTD), 53% of patients with systemic lupus erythematosus (SLE), 42% with Sj?gren's syndrome (SS), and 10% with rheumatoid arthritis (RA). However, this antibody was found neither in the sera from patients with other types of rheumatic diseases nor in healthy volunteers. The screening of this antibody may be useful in diagnoses of MCTD, SLE, and SS. In the present study, we also performed the characterization of the cells reacting with this antibody. The cells proved to be acid phosphatase positive phagocytes in the SRP, that is, red pulp macrophages. Moreover, a histochemical analysis of the reacting antigen in these cells has demonstrated that its antigenic activity is NaIO4 and RNase sensitive, suggesting that the antigen may be associated with RNA.     

Erythrophagocytosis of the lymph node macrophages caused by autotransplantation of the splenic tissue into the lymph nodes of rat

The rat splenic tissue, autotransplanted into the mesenteric lymph node, regenerated about three months. The recipient lymph node became reddish because of the increase of erythrocytes in the lymphatic sinuses and medullary cords. The macrophages in the lymphatic sinus showed an active erythrophagocytosis. In electron microscopy, these macrophages contained the graded degradation of erythrocytes and closely contacted with lymphocytes. These observations represent the splenic autotransplants in the lymph node changes the recipient lymph node into the hemolymph node, and the lymph node macrophages do not recognize the self-erythrocytes as self-ones.     

Murine alpha-2-macroglobulin: localization on a subpopulation of macrophages

We have previously shown that mouse peritoneal macrophages synthesize and secrete alpha-2-macroglobulin (alpha 2M) in culture. We have now examined whether alpha 2M delineates a subpopulation of murine macrophages. Mouse alpha 2M was purified from plasma by gel filtration and immunoabsorption to remove IgM. Purified alpha 2M was an active proteinase inhibitor, had a pI of 4.8, and a relative molecular mass (Mr) of 765 kD which dropped to 194 kD on reduction. Antisera raised against mouse alpha 2M were not cross-reactive with guinea pig or human alpha 2M, or with antigens in guinea pig, human, bovine or equine serum. One to 18 per cent of freshly isolated bone marrow, resident peritoneal or induced peritoneal macrophages displayed immunoreactive cytoplasmic and surface alpha 2M as detected by single cell immunoperoxidase assay or flow cytometry. alpha 2M was not detected in or on thymocytes or bone marrow lymphocytes. In culture, bone marrow-derived macrophages displayed peak levels of cytoplasmic alpha 2M on day 7 and two peaks of surface alpha 2M on days 2-3 and 5-7. The fact that alpha 2M can function as an anti-proteinase, can modulate lymphokine production and is present on inflammatory macrophages suggests a regulatory role for macrophages bearing and secreting this molecule at tissue sites of inflammation.     

轻度热应激大鼠脾脏巨噬细胞BiP蛋白的表达与意义

目的： 探讨轻度热应激脾脏巨噬细胞免疫球蛋白结合蛋白(BiP/GRP78)的表达及其对细胞功能改变的影响。方法: 原代培养脾脏巨噬细胞，将细胞置于41 ℃恒温箱中，使细胞轻度热应激，1 h后恢复到37 ℃，分别检测应激后0 min、30 min、60 min、120 min、180 min巨噬细胞BiP mRNA、BiP蛋白的表达；同时段分别检测巨噬细胞吞噬功能、杀伤活性和趋化作用。结果: （1）轻度热应激后，脾脏巨噬细胞BiP mRNA的表达明显增加，30 min后到达高峰，60 min、120 min时仍高于对照组，180 min后恢复至正常水平。BiP蛋白的表达在60 min后到达高峰，120 min、180 min时仍高于对照组。（2）轻度热应激后，脾脏巨噬细胞的吞噬功能、杀伤活性和趋化作用均明显增强，且与BiP mRNA、BiP蛋白表达的改变基本同步。结论: 轻度热应激脾脏巨噬细胞BiP mRNA、BiP蛋白的表达与巨噬细胞功能发生同步改变，提示BiP蛋白表达上调可能与轻度热应激脾脏巨噬细胞功能增强密切相关。     

DEC-205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.     

Light and electron microscopic observations of the white pulp of the dog spleen during hemorrhagic shock

Structural changes of the white pulp of the spleen were investigated in dogs subjected to hemorrhagic shock. At 1 h after hemorrhage (early impending stage), hydropic changes of the germinal center cells were observed by electron microscopy, while no remarkable change could be found by light microscopy. At 2 h after hemorrhage (end of impending stage), tangible bodies increased in number in the germinal center as a result of destruction of germinal center cells. In addition, tangible bodies appeared also in the perifollicular zone, while there were few tangible bodies in the follicular area. At the end of critical stage (3 h after hemorrhage), destruction of cells was remarkable not only in the germinal center and perifollicular zone, but also in the follicular area. Tangible bodies and tangible body macrophages were abundant in germinal center, follicular area and perifollicular zone. Moreover, acellular areas frequently appeared in the follicular area which was composed predominantly of small lymphocytes. At the terminal stage of normovolemic shock, the cells of white pulp including perifollicular zones were markedly decreased. Hemorrhages were frequently recognized in the germinal center and follicular area.     

Localization of the CB1 type cannabinoid receptor in the rat basolateral amygdala: high concentrations in a subpopulation of cholecystokinin-containing interneurons.

The neuronal localization of the CB1 cannabinoid receptor in the rat basolateral amygdala was studied using peroxidase and fluorescence immunohistochemical techniques. All nuclei of the basolateral amygdala contained a large number of lightly stained pyramidal neurons and a small number of more intensely stained non-pyramidal neurons. Most of the latter cells had medium-sized to large multipolar somata and three to four aspiny dendrites, but some exhibited smaller oval somata. The axon initial segments of some of these non-pyramidal neurons exhibited large swollen varicosities in colchicine-injected animals, suggesting that much of the CB1 receptor protein is transported down the axons of these cells. Double-labeling studies using immunofluorescence histochemistry combined with confocal laser scanning microscopy revealed that the great majority of non-pyramidal neurons with CB1 receptor immunoreactivity belonged to a cholecystokinin-containing subpopulation. Whereas none of the other subpopulations of non-pyramidal neurons (exhibiting immunoreactivity for calretinin, parvalbumin, or somatostatin) expressed high levels of CB1 receptor immunoreactivity, a small percentage of these cells exhibited low levels of immunoreactivity.The results indicate that cannabinoids may modulate the activity of pyramidal projection neurons as well as a subpopulation of cholecystokinin-containing non-pyramidal neurons in the basolateral amygdala. Previous studies indicate that most of the latter are inhibitory interneurons that utilize GABA as a neurotransmitter. The intense staining of the cholecystokinin-containing interneurons and the evidence that large amounts of CB1 receptor protein are transported down the axons of these cells suggests that, as in the hippocampus, cannabinoids may inhibit the release of GABA from the axon terminals of these neurons.