Evidence for the existence of a subsinus layer of the peripheral cortex in the lymph node of the rat

Summary The peripheral cortex of a lymph node consists of folliculo-nodules (follicles with a nodule or germinal center) and an extrafollicular zone. In the course of an analysis of the nodes in rats under various experimental or abnormal conditions, our attention became focused on the particularities of a thin layer of peripheral cortex underlying the subcapsular sinus. The present paper reports on observations which led to the identification of the subsinus layer of the peripheral cortex. The observations suggest that this layer complements the activity of the inner wall of the subcapsular sinus. The layer probably contributes to the selection of certain elements from the afferent lymph and guides them towards the node area where they can join the cell population involved in the immunological activity in which they are to participate.     

Interdigitating reticulum cells in the popliteal lymph node of the rat

Summary Electronmicroscopic and cytochemical studies were performed to localize interdigitating reticulum cells (IDC) in the popliteal lymph node of the rat.The morphological features of the IDC of the rat correspond to those described for other species, but also show similarities to normal macrophages in the rat. This is considered to be an argument in favour of the common origin of IDC's and macrophages.Ultrahistochemical studies with horseradish peroxidase (HRP) reveal no phagocytotic capacity of IDC's. After perfusion fixation containing ruthenium red (RR) the surface coat stains heavily: RR is also found deep in the membrane invaginations of the IDC, indicating the presence of polyanionic sialoglyco-proteins. The post-capillary-venules (PVC) are very permeable to both HRP and RR.The phosphotungstic acid-chromic acid stain (PTA-CrA) also reveals glycoproteins in the surface coat; these glycoproteins are susceptible to -neuraminidase, whereas glycoproteins in the Golgi complexes, lysosomes and in the vesicular complexes of IDC are not. The glycoproteins of the latter are susceptible to 0.1 N NaOH. These findings indicate that IDC produce different kinds of glycoprotein, one of which may be secreted and act as a factor for stimulating peripheral T-lymphocytes.Intimate contact between IDC's and PCV's could be observed. It is therefore conceivable that IDC's play an important role in the homing of T-lymphocytes.     

Diffusion of a lymph-carried antigen in the fiber network of the lymph node of the rat

Summary A lymph-carried antigen is retained preferentially in those areas of the subcapsular sinus of a lymph node overlying the extrafollicular zone of the peripheral cortex. There, it becomes associated with the reticular fibers crossing these particular sinus areas. We wondered whether the antigen thereafter diffuses along the extensions of these fibers which form a peculiar network in the cortical pathways of migration of circulating lymphocytes (CPMCL), leading to the different cell populations effecting the immune responses. Fluorescein isothiocyanate (FITC)-conjugated antigens were injected locally into rats sacrificed 0.5–24 h later. The antigens diffused along the fibers of the CPMCL. It is proposed that this diffusion constitutes one mechanism of stimulation of recruited circulating lymphocytes and of orientation of their migration towards the proper effector-cell population.This work was supported by the Medical Research Council of Canada     

Characterization of dendritic cells,isolated from normal and stimulated lymph nodes of the rat

Summary Non-lymphoid dendritic cells were isolated from normal and paratyphoid vaccine-stimulated lymph nodes draining the rat skin. They were studied using enzymecytochemical, immunocytochemical and electron-microscopical methods. These cells had an irregular outline and an eccentrically situated nucleus. All showed acid phosphatase activity in a central area and expressed Ia antigen on the plasma membrane. Birbeck granules were exclusively present in dendritic cells isolated from lymph nodes in the induction phase of the immune response. This observation concurs with the presence of Birbeck granules in interdigitating cells in situ during the same period of the immune response. It is concluded that the dendritic cells are the in-vitro equivalents of the non-actively phagocytizing population of interdigitating cells.     

The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat

Summary The popliteal lymph nodes were removed from young rats of various ages five days after a single immunization with TNP-KLH in the hind footpads. Cryostat sections of the lymph nodes were investigated by means of enzyme and immunohistochemical techniques at the light-microscopical level.The presence and localization of anti-TNP antibody-containing cells were examined using a new technique to visualize specific antibodies. Moreover, the development of the lymph nodes following exogenous antigenic stimulation was compared with that of unstimulated lymph nodes.Specific antibody-containing cells could not be found before day 15 after birth, in rats immunized at day 10. From that time these lymphoid cells were located primarily at the border between cortex and medulla. Younger popliteal lymph nodes showed only aspecific immunoglobulin-containing lymphoid cells. With age, the number of specific antibody-containing cells tended to increase. These cells were more mature, according to morphological criteria and were located nearer the medulla.The first primary follicles were seen at day 19, as was the case in unstimulated animals. The first secondary follicles, containing germinal centers, were detected at day 23, whereas in unstimulated popliteal lymph nodes they were never found.Trapping of immune complexes could not be demonstrated before day 33 after birth. The later appearance of this phenomenon might be a consequence of the techniques applied to demonstrate specific antibody-containing cells.Abbreviations PLN popliteal lymph node - FDC follicular dendritic cell - IDC interdigitating cell - HEV high endothelial venule - TNP trinitrophenyl - KLH keyhole limpet hemocyanin - PBS phosphate-buffered saline - GCPC germinal center precursor cell - sIg surface immunoglobulin - cIg cytoplasmic immunoglobulin     

Glycosyl receptors in macrophage subpopulations of rat spleen and lymph node

Summary We have developed an immunohistochemical method for the in vivo and in vitro detection of glycosyl receptors in rat spleen and lymph nodes by using neoglycoproteins. The receptor in both organs recognized mannose coupled to bovine serum albumin (mannose-BSA), fuscose-BSA, N-acetylglucosamine-BSA and to a lesser extent glucose-BSA, but not galactose-BSA or N-acetylgalactosamine-BSA. In vitro neoglycoprotein-receptor binding was Ca²⁺ dependent and could be inhibited by mannan but not by mannose. Simultaneous staining with the monoclonal antibodies ED1, ED2 or ED3 revealed that only ED1-and ED3-positive macrophages were involved in the binding of neoglycoproteins. In the spleen, the marginal-zone macrophages and a subpopulation of the marginal metallophils possess glycosylbinding receptors. In the lymph nodes, the medullary sinus macrophages and a subpopulation of the outercortex macrophages are able to bind neoglycoproteins.     

Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens

Hamsters of the randomly bred LAKZ and inbred LSH strains were infected with Dipetalonema viteae, and the in vitro responses of lymph node and spleen lymphocytes to male and female worm antigens and phytohemagglutinin (PHA) were measured by a [³H]-thymidine-uptake assay at various times after infection. The PHA response remained unchanged at the level of controls in infected LAKZ hamsters while LSH hamsters showed a depressed response to the mitogen during late infection. Stimulation of lymph node cells by filarial antigens was maximal in both strains of hamsters at Week 4 postinfection, almost reaching values obtained in PHA stimulated cultures. A similar high lymphocyte transformation reaction was measured after the injection of dead third stage larvae. During transient microfilaremia, when antibody titers reached a maximal level, the lymphocyte reactivity to filarial antigens decreased drastically and only occasionally was demonstrated in hamsters 20 and 30 weeks after infection. No correlation between lymphocyte reactivity and parasitological findings (worm load or intensity and duration of microfilaremia) could be demonstrated. The cellular unresponsiveness to filarial antigens was further analyzed in chronically infected LAKZ hamsters. No suppressor cells could be found in lymphocyte suspensions of nonresponding hamsters. A challenge infection did not restore lymphocyte reactivity. Serum of chronically infected hamsters caused marked inhibition when added to filarial antigen-sensitive lymphocytes. Lymphocytes from hamsters with a mixed D. viteae and Schistosoma mansoni infection responded as well to soluble schistosomal egg antigens at Week 30 of a D. viteae infection as lymphocytes from hamsters infected with S. mansoni alone. The humoral immune response to schistosomal antigens, however, was significantly lower in animals with a mixed infection.     

The postnatal development of cell populations in the rat popliteal lymph node

Summary The postnatal development of the various cell populations in the rat popliteal lymph node was investigated applying enzyme-histochemical and immunohistochemical techniques. From birth, T-lymphocytes and interdigitating cells were demonstrable. During the development of the young lymph node, T-lymphocytes of the helper phenotype outnumbered the T-cells with a suppressor phenotype; they account for approximately 70% and 30% of all T-lymphocytes, respectively. At the very first day of postnatal life, post-capillary venules were already present. B-lymphocytes occurred later than T-cells during ontogeny; they were found on the second day after birth, most of them being IgM- or IgG-bearing lymphocytes. The first primary follicles occurred at day 18 and contained principally membrane-stained IgM cells and, to a lesser extent, membrane-stained IgG cells. The appearance of follicular dendritic cells correlated with the formation of primary follicles. With respect to the macrophages, it appeared that the ED1- and ED3-positive subpopulations were present with a similar distributional pattern as seen in adults, but in considerably lower numbers. The expression of ED2, however, showed a sudden increase in the third week of life. Findings of the present study are discussed in relation to those obtained in other investigations dealing with the ontogenetic development of lymphoid organs.Abbreviations IDC interdigitating cell - cIg cytoplasmic immunoglobulin - sIg surface immunoglobulin - FDC follicular dendritic cell - PBS phosphate-buffered saline - PCV post-capillary venule - PLN popliteal lymph node     

Structure of sinuses in the human lymph node

Summary A casting technique has been employed to display in three dimensions, the lymphatic microcirculation within the human lymph node. The casting compound filled the marginal sinus, and diffusely permeated the cortical lymphoid parenchyma. However, deep within the lymph node in the medullary region, the medium remained within the limits of the sinus walls. The casts showed well-defined channels appearing similar to vessels. These converged into larger vessels, which drained into efferent lymphatics leaving the node at the hilus.Electron microscopic examination showed that the outer wall of the marginal sinus and the trabecular side of trabecular sinuses had an intact, continuous endothelium with a basement membrane. However, gaps were present in the inner wall of the marginal sinus, as well as in the parenchymal wall of the trabecular sinus. In the medulla, the sinuses were lined by endothelial cells which appeared similar to macrophages. The sinus lining was incomplete and possessed numerous perforations. These observations indicated that sinus walls adjacent to connective tissue served as a barrier to cell movement, but those adjacent to a large lymphoid cell population had gaps, with cells in apparent transit between sinus lumen and parenchyma.     

The early postnatal development of the primary immune response in rat popliteal lymph node,stimulated with thymus-independent type-1 and type-2 antigens

Summary To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.Abbreviations KLH keyhole-limpet haemocyanin - LPS lipopolysaccharide - PBS phosphate-buffered saline - PLN popliteal lymph node - TD thymus-dependent - TI thymus-independent - TNP trinitrophenyl     

Ultrastructure and permeability of lymph node microvasculature in the mouse

Summary The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used.All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

The development of innervation in the rat atrioventricular node

Summary The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed.Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day.These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.     

The marginal sinus in the perifollicular region of the rat spleen

Summary The marginal sinus in the spleen of the Wistar rat surrounds the follicle and has more numerous PAS positive fibers on the inner wall than on the outer wall. India ink- and lead oxide-gelatin were injected into the abdominal aorta. It was found that much of the india ink-gelatin accumulated in the marginal sinus, the marginal zone, and part of the red pulp, while most of the lead oxide-gelatin collected in the marginal sinus.Ultrastructurally, the capillaries of the follicle were found to open into the marginal sinus. Regions not perforated by the marginal sinus lie between the follicle and the marginal zone. The wall of the marginal sinus is discontinuous and the discontinuities are wider on the marginal zone side than on the follicle side. The relationship of these findings is discussed.A part of this study was presented at the 64th Annual Meeting of the Japanese Pathological Society, Takatsuki, April, 1975     

Computed tomography-based radiomic model at node level for the prediction of normal-sized lymph node metastasis in cervical cancer

PurposeRadiomic models have been demonstrated to have acceptable discrimination capability for detecting lymph node metastasis (LNM). We aimed to develop a computed tomography–based radiomic model and validate its usefulness in the prediction of normal-sized LNM at node level in cervical cancer.MethodsA total of 273 LNs of 219 patients from 10 centers were evaluated in this study. We randomly divided the LNs from the 2 centers with the largest number of LNs into the training and internal validation cohorts, and the rest as the external validation cohort. Radiomic features were extracted from the arterial and venous phase images. We trained an artificial neural network (ANN) to develop two single-phase models. A radiomic model reflecting the features of two-phase images was also built for directly predicting LNM in cervical cancer. Moreover, four state-of-the-art methods were used for comparison. The performance of all models was assessed using the area under the receiver operating characteristic curve (AUC).ResultsAmong the models we built, the models combining the features of two phases surpassed the single-phase models, and the models generated by ANN had better performance than the others. We found that the radiomic model achieved the highest AUCs of 0.912 and 0.859 in the training and internal validation cohorts, respectively. In the external validation cohort, the AUC of the radiomic model was 0.800.ConclusionWe constructed a radiomic model that exhibited great ability in the prediction of LNM. The application of the model could optimize clinical staging and decision-making.     

Infection-induced regulation of natural killer cells by macrophages and collagen at the lymph node subcapsular sinus

Infection leads to heightened activation of natural killer (NK) cells, a process that likely involves direct cell-to-cell contact, but how this occurs in vivo is poorly understood. We have used two-photon laser-scanning microscopy in conjunction with Toxoplasma gondii mouse infection models to address this question. We found that after infection, NK cells accumulated in the subcapsular region of the lymph node, where they formed low-motility contacts with collagen fibers and CD169(+) macrophages. We provide evidence that interactions with collagen regulate NK cell migration, whereas CD169(+) macrophages increase the activation state of NK cells. Interestingly, a subset of CD169(+) macrophages that coexpress the inflammatory monocyte marker Ly6C had the most potent ability to activate NK cells. Our data reveal pathways through which NK cell migration and function are regulated after infection and identify an important accessory cell population for activation of NK cell responses in lymph nodes.     

Overexpression of LI-cadherin in gastric cancer is associated with lymph node metastasis

Gastric cancer remains the second leading cause of cancer deaths worldwide. Patients usually present late with local invasion or metastatic diseases. The present study investigated the expression level of liver-intestine cadherin (LI-cadherin) by RT-PCR and its correlation with clinicopathological data in 71 pairs of tumor and non-cancerous gastric mucosa. Protein expression level of LI-cadherin was determined by Western blotting and immunohistochemistry. The mRNA of LI-cadherin was highly expressed in tumor as compared to non-cancerous mucosa. Lymph node metastasis was significantly associated with the expression of LI-cadherin (p=0.038). On multivariate analysis, T staging and LI-cadherin expression were found to be independent factors associated with lymph node metastasis.     

Scanning electron microscope observation of the dog mesenteric lymph node

Summary The perfused large mesenteric lymph node of the dog was observed under the scanning electron microscope.The lymph sinus contains reticulum cells which mostly are two-dimensionally formed stellate plates oriented in a uniform direction. Large round macrophages are loosely fixed by the reticulum cell processes. No intermediate type between both cells has been observed. Macrophages having a few long tentacle-like projections are densely covered by clubbed cytoplasmic processes. Smaller round cells, probably plasma cells and lymphocytes also remained in the sinus.The pulp of the node is built up by reticulum cells, much smaller than those in the sinus, and by densely packed round cells including a few macrophages.The trabeculae and the reticulum of the nodal parenchyme form a continuous structure.Cordial thanks are expressed to Dr. Toshihiro Ishii, Professor of Anatomy of the Tohoku University Medical School, for his valuable advice. Thanks are also due to the kind cooperation of Mr. Akira Kubotsu of the Central Research Laboratories, Kuraray Co. Ltd.     

Basement-membrane components associated with the extracellular matrix of the lymph node

Summary Lymph nodes contain an extensive array of extracellular matrix fibers frequently referred to as reticular fibers because of their reticular pattern and positive reaction with silver stains. These fibers are known to contain primarily type-III collagen. In the present study, frozen and plastic-embedded sections of mouse and human lymph nodes were subjected to immunostaining with a panel of monospecific antibodies directed against type-IV collagen, type-III collagen, laminin, entactin, and heparan sulfate proteoglycan. Immunofluorescent staining revealed that, in addition to being uniformly stained with antibodies to type-III collagen, these fibers also stained positively with antibodies to type-IV collagen and to other basement-membrane-specific components. Furthermore, the basement-membrane-specific antibodies stained the outer surface of individual fibers. These same type-III collagen-rich fibers were distinct from blood vascular basement membranes since they did not react with antibodies to factor VIII-related antigen, an endothelial-cell-specific marker. The role of these basement-membrane-specific components associated with the reticular fibers of lymphoid tissue is unknown. However, it is possible that the ligands promote attachment of reticular fibroblasts as well as macrophages and lymphocytes to the extracellular matrix fibers.     

Depletion of macrophages and disappearance of postcapillary high endothelial venules in lymph nodes deprived of afferent lymphatic vessels

Summary The afferent lymphatic vessels of rat popliteal lymph nodes were interrupted, and the histological alterations in the lymph nodes occurring 1 to 14 weeks after operation were studied. One week after operation the number of macrophages was considerably reduced and continued to decrease during the subsequent time periods studied. A 6 weeks most macrophages had disappeared. Simultaneously the immunological activity diminished and had completely disappeared 8 weeks after operation. Three weeks after operation the endothelial cells of the postcapillary high endothelial venules had flattened, and the number of immigrating lymphocytes was greatly reduced. Subsequently the lymph nodes became depleted of both macrophages and lymphocytes, leaving only the reticuloendothelial framework.