Heterogeneity of stromal cells in the human splenic white pulp. Fibroblastic reticulum cells,follicular dendritic cells and a third superficial stromal cell type

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD271⁻ and CD271^+/− stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27⁺ and switched B lymphocytes, but also scattered IgD⁺⁺ B lymphocytes and variable numbers of CD4⁺ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far.     

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.     

Monoclonal antibodies against mouse splenic stromal cells

A panel of rat monoclonal antibodies directed against mouse splenic stromal cells were isolated. These monoclonal antibodies were Immunohistochemically divided into four groups which reacted with non-lymphoid cells of the murine spleen; (1) in the white pulp, (11) at the marginal zone, (111) in the red pulp, and (IV) on the endothelium of splenic blood vessels. These monoclonal antibodies were studied Immunohistochemically In lymphoid organs by means of light and electron microscopy. Monoclonal antibodies SS-4 (group I) reacted with fibroblastic reticulum cells that were distributed only in the white pulp of the spleen and In the follicular areas of lymph nodes. The SS-4 staining cell, In clustered splenic stromal cells, formed colonies which Included a small number of Thy-1 positive lymphocytes. Therefore, we concluded that SS4 staining stromal cells comprise the lymphoid cornpartment. In contrast, monoclonal antibodies SS-1, SS-3 and SS-5 (group II) reacted with dendritic shaped cells in the marginal zone of the spleen. Examination of splenic extra-medullary hematopolesis in mice rescued by bone marrow transplantation after lethal irradiation revealed that SS-3 and SS-5 reacted with dendritic shaped stromal cells in clonal nodules of engrafted marrow in the red pulp. SS-3 and SS-5 staining cells could not be observed in physiologic hematopoiesis of non-transplanted mice. It was suggested that SS3 and SS-5 staining stromal cells are Involved in primitive hematopoiesls. Monoclonal antibodies SS2, SS-6 and SS-7 (group 111) mainly reacted with dendritic cells and macro-phages in the red pulp. Monoclonal antibodies SS-8 and SS-9 (group IV) reacted with endothelial cells of blood vessels and sinuses. These findings of heterogeneity in mouse splenic stromal cells are further evidence that specific micro-envlronments are composed by speclalired stromal cells.     

大鼠脾脏的组织发生

应用光镜、扫描电镜和透射电镜对大鼠脾脏的组织发生进行了形态学观察。脾原基发生在妊娠第16天,继则血管出现,血窦形成,脾脏造血开始。妊娠第19天,网状细胞围绕着发生中的小动脉形成动脉周围鞘。随之淋巴细胞聚集于小动脉周围。出生后,各种血细胞造血相继终止,红、白髓不断发育成熟,生后第5天观察到淋巴树突细胞。边缘带、边缘窦和脾小结相继出现。生后第25天观察到原始生发中心。第40天后,脾脏各部结构基本发育完善。     

Immunohistochemical localization of smooth muscle myosin in human spleen, lymph node, and other lymphoid tissues. Unique staining patterns in splenic white pulp and sinuses, lymphoid follicles, and certain vasculature, with ultrastructural correlations.

The anatomic distribution of smooth muscle myosin, a contractile protein, was determined in a variety of lymphoid tissues (spleen, lymph nodes, tonsils) with the use of highly specific rabbit antibodies to human uterine smooth muscle myosin and an indirect immunoperoxidase technique. In the spleen, in addition to the anticipated immunoreactivity in the walls of arteries, veins, splenic capsule, and trabeculas, other staining patterns were observed. Smooth muscle myosin-containing cells which comprised the adventitia of the trabecular arteries appeared continuous with myosin-containing reticular cells of the white pulp. The latter cells assumed a circumferential pattern within the periarteriolar lymphoid sheaths, then blended delicately with the red pulp at the marginal zone. Ultrastructurally, immunogold techniques demonstrated that smooth muscle myosin in these cells was localized to cytoplasmic filaments. Within the red pulp, a different and distinct staining pattern was observed for the splenic sinuses. Short, regular, orderly, and repetitive bands of immunoreactivity, aligned parallel to the long axis of the sinus, extended between contiguous ring fibers. By immunoelectron microscopy these structures corresponded to distinct bundles of filaments in the endothelial lining cells of the splenic sinuses. Factor VIII associated antigen was also identified in the splenic lining cells in cryostat and paraffin sections, and ultrastructurally. Within the red pulp of the spleen, the sheaths of sheathed capillaries also revealed strong immunoreactivity for smooth muscle myosin. Other sites of immunohistochemical localization of smooth muscle myosin included dendritic reticulum cells present in reactive follicles and in nodular non-Hodgkin's lymphomas. Certain vascular structures, specifically sinus lining cells and Schweigger-Seidel capillary sheaths of the spleen and postcapillary venules of lymph nodes and tonsils, coexpressed smooth muscle myosin and Factor VIII associated antigen. The patterns of localization of smooth muscle myosin are correlated with anatomic structures and possible tissue functions.     

Low-grade B-cell lymphomas of the splenic marginal zone: a clinicopathological and immunohistochemical study of 14 cases

We have studied 14 cases of low-grade, splenic marginal zone, B-cell non-Hodgkin's lymphoma. The clinical presentation in all cases was with splenomegaly and, in 10 cases, circulating neoplastic lymphoid cells in the peripheral blood with involvement of bone marrow. In all cases the splenic white pulp was hyperplastic with expansion of marginal zones and varying degrees of infiltration of germinal centres by neoplastic cells. The cells were a mixture of medium sized lymphocytes with moderate amounts of cytoplasm and finely dispersed nuclear chromatin, together with occasional blast cells with small nucleoli. Satellite red pulp aggregates of tumour cells centred on small epithelioid cell clusters were seen in all cases. These appear to be a characteristic and diagnostically important feature of splenic marginal zone lymphoma. The tumour cells expressed CD20, CD45RA, bcl-2 and the antigens detected by MB2. All cases expressed IgM with light chain restriction. In addition, IgD was expressed in four cases. The follicular dendritic cell network was disrupted in those follicles which were infiltrated by tumour cells. A network of stromal myoid cells, at the periphery of the marginal zone, identified by expression of α-smooth muscle actin, was preserved. Alpha-smooth muscle actin positive dendritic cells were also seen within and around satellite tumour nodules in the red pulp.     

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.     

The splenic red pulp; a histomorphometrical study in splenectomy specimens embedded in methylmethacrylate

The anatomy and pathology of the splenic red pulp was studied in three-dimensional reconstructions of methylmethacrylate embedded blocks of tissue obtained after splenectomy, as well as by morphometrical analysis of a large number of specimens. The sinuses of the spleen form a plexus of anastomosing vessels with remarkable buds. Capillaries end as sheathed capillaries in the cord tissue, the 'filtering' area, but a large proportion of the red pulp cords appear to be 'non-filtering'. These might form part of the lymphatic compartment, which is separate from the white pulp and its extension along the capillaries. This area has not yet been described in man. The change in the volume and structure of the various components of the red pulp were studied in 60 controls and in cases of traumatic rupture, idiopathic thrombocytopenic purpura, aplastic anaemia, autoimmune haemolytic anaemia, congenital spherocytosis, splenic congestion, and Hodgkin's disease. Significant differences were found in the volume of filtering and non-filtering areas, the size of the sinus compartment, and the degree of vascularization; these differences were only partially expected, for instance in disorders with excessive erythrocyte sequestration. A decrease of the 'non-filtering' area in Hodgkin's disease might indicate an unknown aspect of this disease. In agreement with our previous paper on the amount of white pulp, spleens removed because of traumatic rupture and those incidentally removed during abdominal surgery may not be combined as a single control group, because of significant and probably functional differences in the composition also of the red pulp.     

The perifollicular and marginal zones of the human splenic white pulp : do fibroblasts guide lymphocyte immigration?

We investigate the white pulp compartments of 73 human spleens and demonstrate that there are several microanatomical peculiarities in man that do not occur in rats or mice. Humans lack a marginal sinus separating the marginal zone (MZ) from the follicles or the follicular mantle zone. The MZ is divided into an inner and an outer compartment by a special type of fibroblasts. An additional compartment, termed the perifollicular zone, is present between the follicular MZ and the red pulp. The perifollicular zone contains sheathed capillaries and blood-filled spaces without endothelial lining. In the perifollicular zone, in the outer MZ, and in the T cell zone fibroblasts of an unusual phenotype occur. These cells stain for the adhesion molecules MAdCAM-1, VCAM-1 (CD106), and VAP-1; the Thy-1 (CD90) molecule; smooth muscle alpha-actin and smooth muscle myosin; cytokeratin 18; and thrombomodulin (CD141). They are, however, negative for the peripheral node addressin, the cutaneous lymphocyte antigen, CD34, PECAM-1 (CD31), and P- and E-selectin (CD62P and CD62E). In the MZ the fibroblasts are often tightly associated with CD4-positive T lymphocytes, whereas CD8-positive cells are almost absent. Our findings lead to the hypothesis, that recirculating CD4-positive T lymphocytes enter the human splenic white pulp from the open circulation of the perifollicular zone without crossing an endothelium. Specialized fibroblasts may attract these T cells and guide them into the periarteriolar T cell area.     

Disorganization of the splenic microanatomy in ageing mice

The precise mechanisms responsible for immunosenescence still remain to be determined, however, considering the evidence that disruption of the organization of primary and secondary lymphoid organs results in immunodeficiency, we propose that this could be involved in the decline of immune responses with age. Therefore, we investigated the integrity of the splenic microarchitecture in mice of increasing age and its reorganization following immune challenge in young and old mice. Several differences in the anatomy of the spleen with age in both the immune and stromal cells were observed. There is an age‐related increase in the overall size of the white pulp, which occurs primarily within the T‐cell zone and is mirrored by the enlargement of the T‐cell stromal area, concurrent to the distinct boundary between T cells and B cells becoming less defined in older mice. In conjunction, there appears to be a loss of marginal zone macrophages, which is accompanied by an accumulation of fibroblasts in the spleens from older animals. Furthermore, whereas the reorganization of the white pulp is resolved after several days following antigenic challenge in young animals, it remains perturbed in older subjects. All these age‐related changes within the spleen could potentially contribute to the age‐dependent deficiencies in functional immunity.     

High-dose interferon-gamma alters the distribution of B lymphocytes and macrophages in rat spleen and lymph nodes.

Continuous intravenous infusion of rat interferon-gamma (IFN-gamma) for 3 days provokes profound alterations of splenic architecture in LEW rats. The marginal zone of the white pulp is almost totally depleted of B lymphocytes and the follicles are reduced to small remnants. IgM kappa + plasmablasts and plasma cells increase substantially in the outer periarteriolar lymphatic sheath (PALS) and in the splenic red pulp. In addition, marginal metallophilic and marginal zone macrophages are augmented, partially by proliferation. It is discussed whether the activation and proliferation of these macrophages prevent replenishment of the marginal zone and follicles with recirculating B cells. Changes in B lymphocyte and medullary macrophage distribution are also present in submandibular and mesenteric lymph nodes.     

大网膜内植入自体脾组织与原位脾组织的结构比较

目的 :为临床应用自体脾组织植入术提供实验研究资料。方法 :大鼠分为实验组和对照组 ,前者切取 1 /2脾脏去包膜后切成 1mm× 1mm× 1mm大小均匀组织块 ,植入大网膜囊袋内。饲养 6个月后取 2组脾组织制片 ,光镜和电镜定性观察组织结构变化 ,计算机图像分析系统比较血管、红髓、白髓及胶原纤维的面密度 ;免疫组化法结合计算机图像分析测定神经肽 (NPY)阳性神经纤维密度。结果 :神经和边缘窦内皮细胞结构恢复较好 ,血管 ,白髓的面密度值较对照组减少 ,红髓与对照组相当 ,胶原纤维面密度增加。结论 :大网膜内植入的自体脾组织通过再生能恢复脾脏的主要组织结构 ,但不能完全恢复正常。     

自体脾组织移植血供变化的实验研究

目的：探讨自体移植脾组织再血管化的形态构筑特点及其变化规律，同时阐明其血供与功能的关系。方法：采用健康Ｗｉｓｔａｒ大鼠，行脾切除半脾大网膜内移植术，定期采用微血管铸型扫描电镜，观察微血管构筑；通过碳素墨水混悬液灌注后脾组织切片，观察再血管化的演变过程；借助ＭＡＳ图像分析测定微血管的面积分数（Ａａ）。结果：自体脾移植术后１８０ｄ，微血管构筑的主要特征是：中央动脉不够清楚，白髓减少，边缘窦微血管网形态不规则，呈球囊状扩张，红髓内静脉窦扩张尤为显著，形成大的“血管湖”。组织学和图像分析Ａａ观察到移植脾组织呈动态变化，经历一个坏死再生、再血管化的过程。结论：自体移植脾组织的再生及再血管化在术后１８０ｄ基本完成，符合形态与功能相一致的规律。     

Repopulation of lymph nodes and spleen in thymus chimeras after lethal irradiation and bone marrow transplantation: dependence on the age of the thymus

CAP and Lewis rats were thymectomized and received a syngeneic thymus graft followed by lethal irradiation and syngeneic bone marrow transplantation. In three groups (A: recipient 15 months old, thymus graft 3 months old; B: recipient 3 months old, thymus graft 15 months old; C: recipient and thymus graft both 3 months old), we performed an immunohistologic analysis of the splenic white and red pulp and the paracortical zone of the lymph nodes. The repopulation of these regions was demonstrated with monoclonal antibodies that react with Thy-1 positive cells, peripheral T cells, T helper cells, and T non-helper cells. In the splenic red pulp, more Thy-1 positive lymphocytes were found in group B than in group C. The proportion of T lymphocytes and T helper lymphocytes in the region of the periarteriolar lymphocyte sheath of the splenic white pulp was higher when a young thymus was transplanted (groups A and C) than when an old one was (group B). In contrast, in the splenic red pulp, more T lymphocytes were found in group A than in groups B and C. In the paracortical zone of the lymph nodes, this was demonstrable only for group C versus group B. The proportion of T non-helper lymphocytes in the region of the splenic red pulp was higher in group B than in group C. These results indicate that the repopulation of lymph nodes and spleen after transplantation of an old thymus is delayed, quantitatively reduced, and qualitatively different (more T non-helper lymphocytes).     

Deep splenic lymphatic vessels in the mouse: A route of splenic exit for recirculating lymphocytes

A study of pathways of lymphocyte migration through mouse spleen revealed lymphatic channels closely following arteries in trabeculae and white pulp. Because there is no detailed record of the layout of deep splenic lymphatics in the mouse, or other species, we present our observations in this paper, relating our findings to normal migratory pathways of lymphocytes through the spleen. Lymphatics draining the spleen are so inconspicuous that they often are not mentioned in anatomical discussions. The data presented clearly demonstrate (1) the existence and layout of deep lymphatic vessels in the mouse spleen, and (2) that migrating lymphocytes exit white pulp via these lymphatic vessels. CD4⁺ and CD8⁺ T cell subsets migrated proximally along the central artery from distal (dPALS) to proximal periarterial lymphatic sheaths (pPALS) and exited via deep lymphatic vessels that originate there. B cells migrated from dPALS to enter lymphatic nodules (NOD), thus segregated from T cells. B cells then migrated toward and exited via deep lymphatics. The appearance of labelled lymphocytes in lymph coincided with their disappearance from white pulp compart-ments. Labelled T cells were observed in splenic lymphatics as early as 1 hr after intravenous infusion but took, on average, about 6 hr. B cells took somewhat longer. Thus T and B cells entered and left white pulp through shared pathways, but took divergent intermediate routes through dedicated zones, pPALS for T cells, NOD for B cells.     

Splenic autotransplantation in nude mice. The formation of white pulp

Splenic fragments were autotransplanted into the greater omentum of nude mice (nu/nu) and normal mice to investigate the formation of white pulp in splenic regeneration. The formation of white pulp began from day 5 after transplantation in both groups. Two patterns were found. In one pattern, follicles containing only B cells were first formed and developed into white pulp with poorly developed periarterial lymphoid sheaths (PALS). Most of the white pulp in nude mice and about one third of white pulp formed at day 28 in normal mice followed this pattern. In the second pattern, PALS were first formed and afterwards follicles appeared in the periphery of PALS. This was mainly seen in normal mice. Thus splenic autografts in normal mice contain white pulp with poorly developed PALS, and the homing of T cells begins later than that of B cells. This suggests the possibility of incomplete cellular immunity.     

Stimulation of posttraumatic regeneration of the rat spleen under gravitation overloading

Posttraumatic regeneration of the rat spleen was studied after hemiresection of the organ under gravitation overloading (11 units) when spleen tissue extracts prepared by V. P. Filatov's formula were used as the stimulator. Under gravitation overloading the splenic nodules were smaller in size and smoother in outline, infiltration of their red pulp by lymphocytes was increased, the number of labeled cells and the intensity of label in the reactive centers of the splenic nodules were reduced, and capsule formation in the zone of resection was slowed down. As a result of the use of the stimulator the normal structure of the white pulp was restored, the number of labeled cells was increased, and capsule formation was more rapid. By using the tissue extract under gravitation overloading the process of regeneration (as regards the character and times of development of tissue differentiation) was brought more in line with the ordinary course of posttraumatic regeneration of the spleen.Department of Histology and Pathological Anatomy, Erevan Zooveterinary Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 77–80, January, 1977.     

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.     

神经营养素在小鼠脾的定位研究

为了解神经营养素与免疫系统的关系，用免疫组织化学方法对神经营养素包括神经生长因子（ＮＧＦ）、脑源性神经营养因子（ＢＤＮＦ）、神经营养素３（ＮＴ－３）进行小鼠脾的定位研究。结果表明：３种神经营养素的免疫反应均存在于脾内，但分布特点各不相同。ＮＧＦ主要分布于白髓动脉周围淋巴鞘（ＰＡＬＳ）外层、边缘区（ＭＺ）和红髓（ＲＰ）的巨噬细胞样和淋巴细胞样细胞；ＢＤＮＦ除具有与ＮＧＦ相似的分布特点外，还见于脾淋巴小结生发中心的淋巴细胞样细胞；ＮＴ－３则存在于白、红髓的网状细胞样细胞。这一结果提示，脾的免疫和非免疫细胞均可能产生神经营养素，并提示不同类型的神经营养素对免疫系统有不同的作用。