Acetylcholinesterase (AChE)-positive innervation of the guinea-pig spleen

The presence and intraorgan distribution of the acetylcholinesterase (AChE)-positive nerve structures in the guinea-pig spleen were studied by means of the direct thiocholine method. Visualized AChE-positive nerve fibres entered the guinea-pig spleen at its hilum in the vicinity of the splenic artery branches and intra parenchyma were gradually distributed to form thicker periarterial nerves and also fine adventitial nerve plexus. In described topography the AChE-positive nerve fibres were identified in association with the central artery running through the white pulp. Some of the perivascular nerve fibres associated with the central artery extended away and passed into the periarterial lymphatic sheath (PALS) to reach the marginal zone and in continuation entered into the mantle zone of lymphatic follicles. Several AChE-positive nerve fibres were seen in the red pulp but less in the splenic capsule. We did not find any AChE-positive nerve cells in the guinea-pig spleen.     

Lymphocyte circulation in the spleen: Marginal zone bridging channels and their possible role in cell traffic

Histological evidence is presented for distinct, anatomically determined pathways in the spleen for cells in transit between the white pulp and the red pulp prior to entering the draining veins. In rats and mice these appear as narrow channels of lymphocytes which run between both the periarteriolar lymphatic sheath and the red pulp sinuses, and the peripheral white pulp and the red pulp sinuses, crossing the marginal zone in association with fine argentophilic fibres. These marginal zone bridging channels were found to contain labelled T or B cells 4 and 8 hours after injection which suggested that transit was occurring in the direction from white pulp to red pulp rather than the reverse.

Additional histological evidence is given to suggest that, after antigenic stimulation, germinal centre dissociation occurs by release of the germinal centre cells towards the periarteriolar lymphatic sheath before they are shed into the red pulp through marginal zone bridges occurring in the periarteriolar region.

The data are incorporated into a scheme of unidirectional lymphoid cell flow through the spleen. This proposes that the spleen is composed of many functionally discrete units in which the anatomical matrix, reflected by the reticulin fibre pattern, plays a major role. It further implies that the periarteriolar region of the spleen is not totally thymus dependent.

     

免疫应答中小鼠脾GAP-43神经支配的可塑性--神经纤维数量和分布的变化

为了解脾交感神经在免疫功能增强时的形态变化，用免疫组织化学方法观察了ＢＡＬＢ／ｃ小鼠脾内生长相关蛋白样免疫反应（ＧＡＰ－４３－ＬＩ）性神经在结核菌素衍生蛋白（ＰＰＤ）诱发的免疫反应高峰期数量和分布的变化。结果显示，动物在接受两次ＰＰＤ注射（２１ｄ＋７ｄ）后，脾ＧＡＰ－４３－ＬＩ神经纤维密度明显增加并伴有分布和形态的变化。于对照动物，脾ＧＡＰ－４３－ＬＩ神经纤维主要分布于血管周围，少量伸入动脉周围淋巴鞘的脾基质中，而在免疫刺激的动物，不仅血管周围的神经纤维明显增多，动脉周围淋巴鞘外层、边缘区和红髓等免疫应答活跃的部位也出现许多神经纤维。神经纤维分支和纤维上膨体明显增多。以上结果提示：免疫应答中脾神经成分发生活跃的结构重塑，这些变化有可能与神经的免疫调节功能有关。     

Species variation in the structure and function of the marginal zone—an electron microscope study of cat spleen

The marginal zone in the cat spleen consisted of a characteristic mixture of lymphocytes and other blood cells located mainly between the several layers of circumferential reticulum around white pulp. A region of fine-meshed reticulum between white pulp and red pulp, as present in some species, was absent from the cat spleen. Arterial capillaries to the marginal zone were few. Some were continuations of white pulp capillaries, whereas others were red pulp capillaries that likely were continuations of axial capillaries of periarterial macrophage sheaths (PAMS) (ellipsoids). Blood cells deposited in the marginal zone could reach red pulp by passing through the numerous openings in each layer of circumferential reticulum. Lymphocytes appeared to migrate across the marginal zone both toward and away from white pulp. Macrophages lying on the circumferential reticulum of the marginal zone phagocytized cells but did not ingest Thorotrast, although it coated their surfaces. Because of the scarcity of arterial endings and the lack of a macrophage-charged reticular meshwork, the marginal zone in cat spleen is not a major site of blood clearance and phagocytosis. These functions are better served in PAMS and red pulp.     

降钙素基因相关肽免疫反应神经纤维在大鼠脾的分布

用免疫组织化学ＡＢＣ法，研究了降钙素基因相关肽免疫反应神经纤维在大鼠脾内的分布，在大鼠脾内有丰富的ＣＧＲＰ免疫反应神经纤维，它们多呈串珠状，主要分布于红髓。尤其是脾血窦和一些小血管，也见于边缘区的淋巴组织中，偶见于动脉周围淋巴鞘，脾内ＣＧＲＰ免疫反应神经纤维与淋巴细胞关系密切，可能参与调节脾淋巴细胞的发育和功能。     

The human spleen; a histological study in splenectomy specimens embedded in methylmethacrylate

In a series of 316 surgically removed spleens, a histological and supportive immunohistological study was performed on methylmethacrylate sections. The structure of the human white and red pulp differs from the rat spleen in many respects, e.g. the human lacks the marginal sinus and the architecture of the periarteriolar lymph sheath seen in the rat. In man, the lymphoid compartment is in both white and red pulps. In the white pulp separate periarteriolar T-cell areas contain a large lymph-vessel plexus, which was reconstructed in serial sections. The circulation in the red pulp is discussed. The area between the red and white pulp, the perifollicular zone, is not the equivalent of the marginal sinus in the rat. Its anatomy in man suggests that it is an area formed from red pulp during the expansion of new follicles. The micro-anatomy was analysed in 119 controls. In cases of traumatic rupture the white pulp showed evidence of stimulation. A pathognomonic histological picture was not found in idiopathic thrombocytopenic purpura. In haemolytic anaemia the pulp cords were engorged by erythrocytes accompanied by a decreased B/T cell ratio in autoimmune haemolytic anaemia and by an increased B/T cell ratio in congenital spherocytosis.     

Splenic white pulp and associated vascular channels in chicken spleen

We have studied the chicken spleen by light and transmission electron microscopy. Germinal centers are located at the beginning of the central artery which is surrounded by the periarterial lymphatic sheath (PALS). The central artery has no branch crossing the PALS, and there is no histologically identifiable marginal zone in the chicken spleen. The central artery continues as penicilliform capillaries. The mid-portion of the penicilliform capillary is surrounded by the ellipsoid or Schweigger-Seidel sheath. The endothelial lining of this part of the pencilliform capillary contains small channels, formed between neighboring endothelial cells, which enter the ellipsoid. These channels allow circulating substances to accumulate in the ellipsoid. The cells of the ellipsoid are reticular cells, round or ovoid in shape, exhibiting a limited number of cell junctions. At the surface of the ellipsoid are ellipsoid-associated cells (EAC) which have an affinity for toluidine blue. After perfusion fixation, the majority of the cells in the ellipsoid are lost; this suggests weak cellular connections between the ellipsoid cells. The ellipsoid-associated cells remain in loco. On the basis of their shape and cytological features, two stages of EACs can be distinguished. The round or ovoid EACs elaborate active Golgi zones surrounded by numerous small vesicles containing an electron-dense substance. Occasionally, mitotic figures can be seen among them which may suggest that they are immature forms. In the second or more mature stage, the EACs assume an elongated spindle shape. The spindle-shaped EACs reveal inactive Golgi cis-ternae and fewer but larger granules than the round or immature EACs. Unmyelinated nerve fibers and nerve ending were observed in the ellipsoid. Perfusion fixation reveals that the distal portions of the penicilliform capillaries extend into the red pulp and become the sinuses. Therefore, the circulation of the chicken spleen is anatomically closed except for the channels in the mid-portion of the penicilliform capillary. The periellipsoid white pulp (PWP) possesses a few small lymphocytes, macrophages, and plasma cells with the majority of cells being young blastlike cells which may replenish the EACs and ellipsoidal cells. Carbon, injected intravenously, appears on the EAC membrane by 30 minutes, while Brucella abortus is completely phagocytized at this time. Binding the carbon triggers the EAC to detach from the ellipsoid and migrate to the periellipsoidal white pulp (PWP). The EACs phagocytize carbon by 2 hours, and by 5 and 8 days are present in the red pulp and surround the germinal centers at the border of the periarterial lymphatic sheath, respectively. Secretory cells which have been identified in germinal centers might originate from EACs.     

The differentiation of white pulp and red pulp in the spleen of human fetuses (72–145 mm crown-rump length)

The development of the white and red pulp in spleen from thirteen human fetuses measuring from 72 mm to 145 mm in crown-rump length (CRL) was studied using the electron microscope. This period follows the development of the primary vascular reticulum (Weiss, '73). The white pulp appears first as a periarterial sheath with variable numbers of large and medium-sized lymphocytes, monocytes, macrophages, and some granulocytes and erythrocytes. It is always rich in macrophages. At 90 to 100 mm CRL, reticular cells closely associated with collagen and having a distinctive dark hyaloplasm appeared first in the endothelium and close about blood vessels and then out in the pulp. In the white pulp they became circumferentially arranged about the central artery while in the red pulp they formed a branching reticulum. Small lymphocytes were present in increasing number in the periarterial lymphatic sheath after the development of the circumferential reticulum. The venous sinuses developed and the marginal zone stood out as an erythrocyte-rich and macrophage-rich shell of reticulum surrounding the periarterial sheath.     

Migration of macrophages from the marginal zone to germinal centers in the spleen of mice

Histological observations of the mouse spleen were carried out at different times after intravenous carbon injection. Large carbon-laden macrophages appeared in great numbers in the marginal zone soon after injection. They came together favorably around the germinal centers. Possible migration of these cells toward the germinal centers diffusely from the periphery of the white pulp or through the periarterial lymphoid sheath was suggested. These macrophages entered the germinal centers on a large scale and clustered for a long period--at least 180 days. Since the same type of macrophages were observed persistently in the marginal zone, it was thought that some of them might arise from the blood stream. Possible migration of these cells from the marginal zone toward the germinal centers was also persistently observed. A second type of much smaller carbon-laden macrophages was seen in the white pulp. However, they never showed any favorable localization in the germinal centers as did large carbon-laden macrophages.     

Histogenesis of splenic lesions in Hodgkin's disease.

Histochemical markers were used to identify the various cellular and structural components of the human spleen, and to investigate the histogenesis of the splenic lesions of Hodgkin's disease. The early lesions appear in areas near the central artery (periarterial lymphatic sheath) in the white pulp. The white pulp becomes hypertrophic. The lesions enlarge, extend into the red pulp, and compress the sinuses and the cords of Billroth. The derivations of various "histiocytes" contained with the lesions are differentiated by using cytochemical stains for lysosomal enzymes and for granulocytes. The epithelioid cells in the granulomas are rich in those lysosomal enzymes typically seen in phagocytic histiocytes, suggesting that they are indeed true histiocytes. The malignant "histiocytes," including the mononuclear Hodgkin cells, the binucleated Sternberg-Reed cells, and the multinucleated giant cells, do not contain significant amounts of lysosomal enzymes and more closely resemble stimulated lymphocytes. The splenic lesions in Hodkin's disease may be the result of a lymphocytic and histiocytic cellular response to an unknown agent, which reaches the spleen through the central artery in the white pulp.     

GAP-43免疫反应神经纤维在小鼠脾脏的分布及其性质的研究

用免疫组化方法研究了GAP－43免疫反应阳性神经纤维在成年小鼠脾脏的分布及其性质。结果显示，GAP43样免疫反应阳性纤维不仅广泛分布于脾内血管周围，还存在于白髓、红髓的淋巴细胞之间。应用6-羟多巴（6－OHDA）损毁儿茶酚胺能神经后，TH、NPY免疫组化染色不能显示任何阳性神经纤维，而GAP－43免疫反应阳性神经纤维尚有部分存留．它们多位于较大的血管周围，较直且平滑、膨体少。此结果表明，成熟小鼠脾内绝大部分单胺能神经纤维和少部分非单胺能神经纤维含有GAP－43，是具有可塑性的神经纤维。     

The species-specific structure of microanatomical compartments in the human spleen: strongly sialoadhesin-positive macrophages occur in the perifollicular zone, but not in the marginal zone.

The microanatomical structure of human and rat splenic white pulp is compared, with special emphasis on the localization of the marginal zone occupied by immunoglobulin M (IgM)+ IgD-/dull B lymphocytes and its specialized macrophages. Our study reveals that in contrast to rats, the marginal zone of humans primarily exists in the vicinity of primary and secondary splenic follicles and that it is almost absent around the periarteriolar T-cell zones. We demonstrate that in humans there is an additional compartment, the perifollicular zone, located between the marginal zone and the red pulp. The perifollicular zone is a dynamic region of variable cellular and phenotypic composition, which can be regarded either as a part of the red pulp or of the follicles. In most cases the perifollicular zone appears as a compartment of the red pulp containing erythrocyte-filled spaces which differ from the typical red pulp sinusoids. Similar to the splenic cords, the perifollicular zone mostly harbours scattered B and T lymphocytes. However, sometimes B lymphocytes clearly predominate in the perifollicular area. In addition, strongly sialoadhesin-positive macrophages form sheaths around capillaries in the perifollicular zone. Such capillary sheaths are not observed in rats. In humans weakly sialoadhesin-positive macrophages are also present in the perifollicular zone and in the red pulp. In some specimens sialoadhesin is, however, strongly expressed by a large number of dispersed perifollicular macrophages. Interestingly, in striking contrast to rats, the human marginal zone does not contain sialoadhesin-positive macrophages and marginal metallophilic macrophages are also absent in humans. Thus, sialoadhesin-positive macrophages and IgM+ IgD- memory B lymphocytes both share the marginal zone as a common compartment in rats, while they occupy different compartments in humans. We show that the human splenic marginal zone does not contain a marginal sinus and assume that in humans the perifollicular region is the compartment where antigen and recirculating lymphocytes enter the organ.     

中华鳖脾脏椭球的微细结构及其循环特征

目的 观察和分析12只中华鳖脾脏椭球的显微和亚显微结构,阐明其循环特点。 方法 应用光镜和透射电镜技术,结合心脏注射墨水悬液方法,显示脾脏椭球的组成、结构与循环。 结果 中华鳖脾脏白髓由动脉周围淋巴鞘（PALS）和椭球周围淋巴鞘（PELS）两种结构组成,缺乏淋巴小结。红髓包括脾索和脾窦,未发现边缘区。中央微动脉穿出PALS后,呈笔毛状分成数条椭球毛细血管,后者周围由PELS包绕。椭球毛细血管的末端直接开口于红髓的脾索,血流注入脾索,而后穿过脾窦内皮间隙进入脾窦。不同于普通血管内皮,椭球毛细血管内皮一般为立方状,基膜不完整,其外即为椭球结构。椭球壁由支持细胞、椭球相关细胞和网状纤维等构成。常见淋巴细胞和红细胞穿过椭球壁。注射墨水悬浮液后40min,可见整段椭球壁上分布着大量墨水碳粒。 结论 中华鳖脾脏椭球毛细血管相当于哺乳动物的高内皮后微静脉,是淋巴细胞和血细胞进出淋巴组织的重要通道。中华鳖脾脏循环属于开放式循环。     

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.     

In vivo homing of thymus-enriched bone marrow cells

A population of adult CBA/J mouse bone marrow (BM) cells enriched by in vitro migration to supernatant prepared from neonatal thymus was labeled with a DNA-binding fluorochrome, Hoechst dye No. 33342 (H33342). Labeled cells were injected into irradiated recipients in order to compare the in vivo localization of the migration-enriched BM (MEBM) cells to the localization of injected nonenriched BM (NEBM) cell controls. A characteristic difference in the distribution of localized cells was observed in the spleen but not in other lymphoid organs. At 2 hr after injection the MEBM cells were located in the marginal zones surrounding the periarterial lymphoid sheaths (PALS) of the splenic white pulp. At 6 hr after injection the MEBM cells were seen distributed between marginal zones and the PALS and by 16 hr they had localized almost exclusively in the white pulp. In contrast, the NEBM cells were located in the marginal zones or red pulp for the duration of the experiment. These observations show that the MEBM cells home selectively to T-cell areas of the spleen. Direct immunofluorescent monoclonal antibody staining of H33342-labeled cells obtained from the recipient spleens at 16 hr demonstrated that the MEBM cells were negative for Thy-1 antigen, indicating that acquisition of Thy-1 was not prerequisite to the observed homing. The results are compared to known localization patterns of mature lymphocytes.     

Effects of cyclosporin A on the splenic tissue of rats: a histomorphometric analysis

Histological studies demonstrated that long-term cyclosporin A treatment of nonantigenically challenged (untransplanted and unimmunized) Lewis rats markedly reduces the total percentage of splenic white pulp when compared to untreated control spleens (mean = 24 vs 34%, P less than 0.001). Direct measurements of periarteriolar sheaths and marginal zones demonstrated a marked reduction in size of these compartments in cyclosporin A-treated rats compared to untreated controls (P less than 0.001). In addition, there was a striking reduction in cellular density of the periarteriolar sheaths (P less than 0.001) and a minimal reduction in cellular density of the marginal zones (P less than 0.1) in the cyclosporin A-treated group when compared to untreated controls. There was no significant difference in total splenic size between the cyclosporin A-treated and the control groups, as indicated by total cross section measurements (mean = 33.3 vs 35.0, P less than 0.4). Qualitative observations of methyl green-pyroninophilic cells within and surrounding the marginal zones of the cyclosporin A-treated spleens revealed a much greater proportion of large pyroninophilic lymphocytes, which suggests that they are B immunoblasts. We conclude that long-term cyclosporin A treatment depletes splenic periarteriolar sheaths and marginal zones, compartments known to contain primary T lymphocytes, and induces an immunoblastic cell proliferation within the marginal zones and red pulp as well.     

Characterization of stromal cells with myoid features in lymph nodes and spleen in normal and pathologic conditions.

Stromal cells with myoid features were identified in rat or human lymph nodes and spleen in normal and pathologic conditions, using antibodies to desmin, alpha-smooth muscle actin, and smooth muscle myosin. In normal lymph nodes, myoid cells (MCs) were present in the superficial and deep paracortex as well as in the medulla, and absent in lymphoid follicles. In the spleen, they were numerous in the red pulp, less abundant in periarteriolar lymphocyte sheaths of the white pulp, and absent in lymphoid follicles. On double immunostaining, alpha-smooth muscle actin and smooth muscle myosin were coexpressed with desmin only in the deep paracortex and parafollicular areas of the lymph nodes, as well as in the MCs of the periarteriolar lymphocyte sheaths and marginal zone of the spleen; the remaining MCs expressed only desmin. When examined by means of electron microscopy, MCs showed a dendritic shape and cytoplasmic bundles of microfilaments with dense bodies scattered between them. When compared with normal conditions, MCs showed changes of distribution and number in several pathologic situations. Additional findings were 1) staining of pericytes surrounding high endothelium venules of lymph nodes with alpha-smooth muscle actin antibodies in man and rat and with desmin antibodies in rats; 2) staining of endothelial cells in these venules with desmin antibodies in rats. It is concluded that a subset of reticular cells in lymph nodes and spleen, as well as pericytes and endothelial cells in high endothelium venules display cytoskeletal features suggesting a myoid differentiation and function.     

The periarteriolar lymphocyte sheath in immunodeficiency T- or B-lymphocyte area?

T- and B-lymphocyte populations in peripheral lymphoid tissues occur in distinct compartments (e.g., the periarteriolar lymphocyte sheath of the splenic white pulp is a T-cell area). The authors report on two patients with severe combined immunodeficiency (SCID) and one patient with immunodeficiency after anti-T-cell treatment for rejection of a heart transplant, in which the area surrounding the central arteriole in spleen white pulp was well-populated despite T-cell deficiency (documented by, for example, severe depletion of lymph node paracortex). Immunologic phenotyping showed the B-lymphoid lineage of lymphocytes at this location. The framework in the periarteriolar area consisted of follicular dendritic cells, which are typical framework components of B-cell areas. We conclude that assessment of only conventional histopathology of the spleen in these patients leads to erroneous conclusions about the type of immunodeficiency and that immunologic phenotyping is required to document the exact nature of the deficiency.