ROLE OF GERMINAL CENTER IN IMMUNE RESPONSE ENZYME IMMUNO-HISTOCHEMICAL STUDY USING HORSERADISH PEROXIDASE

Using enzyme Immuno-hlstochemlcal method with horseradish peroxidase (HRP), a study on the relation between localization of antigen and antibody, and development of germinal center in lymph node after primary and secondary stimulation was made.
Blastic antibody forming cells and intercellular localization of the antibody were noticed in newly formed germinal centers of draining lymph nodes within 7 days after primary antigenic stimulation. With increase of circulating antibodies produced by the draining lymph nodes, it accumulated in ready made germinal centers of generalized non-draining lymph nodes. At the time of primary stimulation, the HRP antigen was never observed in solid follicles or newly formed germinal centers, and even in ready made germinal centers it became localized only transiently and was never retained. After secondary stimulation, the antigen accumulated quickly in the light zone of germinal centers, being trapped by the previously present antibody. Three days later, antibody forming cells began to be noticed in germinal centers as well as in medullae of non-draining lymph nodes where no antibody forming cells had been so far observed, and in medullae of draining lymph nodes they increased markedly. Passively injected anti-HRP antibody accumulated also in germinal centers of non-primed mice and successively injected antigen was easily trapped in these germinal centers. Passively injected soluble antigen-antibody-complexes were localized in germinal centers of non-primed mice. In both these cases, antibody forming cells appeared only in germinal centers and none in medullae. Blastic cells, proliferating in germinal center and producing 2-ME sensitive antibody were not considered to be directly related with antibody forming cells in the medulla of lymph node.     

ENZYME HISTOCHEMICAL PROPERTY OF HUMAN LYMPH NODES – ITS IMMUNOLOGICAL INTERPRETATION –

Lymph nodes surgically removed from patients with various diseases were histochemically investigated. Enzymes examined were alkaline phosphatase (A1P), acid phosphatase (AcP), β-glucuronidase (β-G), and N-acetyl-/3-glucosaminidase (NGa). On the other hand, adult albino rabbits and Wistar rats were immunized by horseradish peroxidase (HrP) mixed with Freund's complete adjuvant and the localizations of these enzymes, antigen HrP and its antibody in their regional lymph nodes at various stages of the immunization were investigated.
AIP in the human lymph nodes was mainly localized in the reticulum cells of the paracortical areas in a characteristic network pattern. This pattern was most prominent in the lymph nodes of SLE patients with elevated serum y-globulin. In the regional lymph nodes of the albino rabbits immunized with HrP, both A1P and anti-HrP antibody were localized along the processes of reticulum cells in the paracortical areas showing a similar network pattern.
The β-G activity in the human lymph nodes was very intense and particulated in plasma cells and lymphocytes In the medullary cords and paracortical areas. The proliferation of these β-G positive plasma cells and lymphocytes in the medullary cords and paracortical areas was most remarkable In SLE patients. In addition, the β-G activity in these cell types responded most readily and steadily to the antigenic stimulation of HrP to both albino rabbits and Wistar rats.     

AN ULTRASTRUCTURAL STUDY OF THE PRIMARY FOLLICLE IN THE LYMPH NODE

The present authors have studied the cellular composition of the primary follicle (PF) in the germ-free rat lymph nodes and the development of PF in the human lymph node anlages by using the light-and the electron microscopy. According to the ultrastructural study, the cellular composition of PF in the germ-free rat lymph nodes are classified into five cell types, 1) lymphocytic cells (small and medium-sized lymphocytes), 2) large lymphocytes (=large germinal center cells), 3) reticulum cells with desmosome (RED), 4) desmodendric cells (DDC), and S) histiocytes. The interstitial cells with desmosome are classified into DDC and RED based on the relationship to collagenous fibers and the morphological differences. There are amorphous electron-dense intercellular materials among the cytoplasmic processes of DDC, which are distinguished from histiocytes based on the morphological differences. PF emerges as a spherical aggregation of young DDC straight underneath the marginal sinus in the outer cortex of the human embryonal lymph node anlages on the 16th fetal week.     

MSH27抗原在小鼠睾丸中的定位

目的　ＭＳＨ２７是与精、卵质膜融合相关的针对精子抗原的单克隆抗体,本研究检测了此抗体所识别的睾丸抗原分子及其分布。　方法　用免疫亲和层析和聚丙烯酰胺凝胶电泳纯化、分析了ＭＳＨ２７ 识别的小鼠睾丸抗原,并用免疫组织化学和免疫电镜进行了定位。　结果　单抗ＭＳＨ２７ 可识别睾丸提取蛋白中分子量为１２６ｋＤ和１１６ｋＤ的抗原分子,此抗原在睾丸的精原细胞至球形精子细胞的胞质内部均有表达,分布于内质网囊泡内或囊泡膜上。在精子形成过程中,此抗原由胞质内均匀分布变为聚集于顶体帽旁侧的区域,这一区域将形成顶体后区。　结论　１２６ｋＤ和１１６ｋＤ蛋白是单抗ＭＳＨ２７ 在睾丸中识别的抗原分子,它们可能是单抗ＭＳＨ２７在成熟精子中识别的３９ｋＤ抗原的前体分子。     

Sinus-lining cells of the lymph nodes recognized as a dendritic cell type by the new monoclonal antibody Ki-M9.

In the immunobiological characterization of lymph node cells, sinus-lining cells (SLCs) have been given little attention mainly due to the difficulties in their recognition. Ki-M9 is a new monoclonal antibody (MAb) selected for its unique capability to visualize SLCs in human lymph nodes. The details were established by light and electron microscopy and immunoprecipitation of the corresponding biosynthetically labeled antigen. Ki-M9 recognizes a 70-kd protein localized on the surface membrane of SLCs. In the lymphoid tissue, a mild reactivity was exclusively encountered on follicular dendritic reticulum cells in the germinal centers of secondary lymphoid follicles. In other organs, some squamous epithelial and myoepithelial cells were recognized by this antibody. Immunomonitoring of SLCs on light and electron microscopic levels revealed their dendritic morphology, lack of phagosomes, and their close association with type IV collagen fibers. Considering the occurrence of typical dendritic SLCs on the front line of antigen flood, we propose that SLCs be investigated for a possible antigen-binding property.     

HISTO- and IMMUNOPATHOLOGIGAL FEATURES OF TERMINAL AIDS. An Autopsy Case of a Japanese Man with Neurological Signs as Initial Symptoms

An autopsy case of a 37-year-old Japanese man, confirmed as an AIDS patient infected by an undetermined route of transmission, is presented. The initial symptoms of full-blown AIDS in this case were neurological, and the patient died of severe pneumonia 9 months after onset. The main histo- and immu-nopathological features were a marked depletion of helper-inducer T cells and dendritic reticulum cells in the lymphoid tissues, opportunistic infections, and some neuropathology changes. Very few cells, possibly macrophages, immu-noreactive with a monoclonal antibody (VAK-5) against HIV-gag protein P24 were found in the mediastinal lymph nodes. Numerous pathogens had induced opportunistic infections in many organs: severe and generalized cytomegalovirus infection, Pneumocystis carinii pneumonia, bronchopneumonia (possibly due to Pseudomonas aeruginosa) , candidiasis in the tongue and oral cavity, and atypical mycobacteriosis in the pulmonic hilar lymph nodes. Vascular proliferation was found in the perinodal regions of some lymph nodes, but this was not neoplastic vascular proliferation compatible with that of localized Kaposi's sarcoma. ACTA PATHOL JPN 38: 1313-1327, 1988.     

Immunohistochemical localization of Forssman glycosphingolipid-positive macrophages and reticular cells in murine lymphoid tissue

Forssman (Fo) glycolipid antigen, as detected by a monoclonal antibody (mAb), is expressed by a subpopulation of murine macrophages in the spleen and peripheral lymph nodes. The histological distribution of Fo antigen in spleen and lymph nodes was studied by immunostaining of cryosections, and was compared with the staining pattern of four other mAbs known to recognize macrophage subpopulations: F4/80, Mac-1, MOMA-1, and ERTR-9. Fo+ macrophages were found exclusively in the red pulp of the spleen and the medulla of inguinal and axial lymph nodes. Macrophages in the other lymphoid organs were Fo-. Besides macrophages, reticular cells in T-dependent areas of spleen and lymph nodes were Fo+. Attempts to grow colonies of Fo+ macrophages from either bone marrow or spleen precursors were negative. While the usual number of F4/80+ colonies was obtained, only a few, small clusters of Fo+ macrophages were formed, which speaks against an early commitment of precursors to express Fo.     

Breakdown of tolerance to the intestinal bacterial flora in inflammatory bowel disease (IBD)

In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen-presenting cells (APC) and their location. APC, identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania antigen containing Langerhans cells were found in the epidermis, dermis and the regional lymph nodes. We believe these cells translocate from the epidermis to the dermis, where they take up antigen and migrate to the paracortex of the regional lymph nodes. There they are intimately associated with cells of the paracortex, and could be involved in the generation of Leishmania-specific T memory cells. LFA-1 -positive T cells of the CD45RO phenotype were found in the skin lesion. Venular endothelium in the skin lesions expressed intercellular adhesion molecule-1 (ICAM-1), which is the ligand for LFA-1. The migration of lymphocytes from the vascular lumen to the site of inflammation is possibly a result of the interaction of these two adhesion molecules.     

Newly produced virgin B cells migrate to secondary lymphoid organs but their capacity to enter follicles is restricted

The migration of recirculating B cells was compared with that of newly produced virgin B cells following passive cell transfer between congenic strains of rats differing in their kappa immunoglobulin light chain (kappa) allotype. The number and location of donor B cells in the secondary lymphoid organs was determined at intervals following transfer by immunohistology using monoclonal antibodies specific for rat kappa allotypes. Recirculating B cells were obtained from thoracic duct lymph while bone marrow from rats depleted of recirculating cells was used as a source of newly produced virgin B cells. B cells from both sources gained immediate access to extrafollicular areas of secondary lymphoid organs rich in interdigitating cells and T cells. However, in lymph nodes extrafollicular B cells were found adjacent to lymphatics and not in the central paracortex. By 8 h after transfer most B cells from thoracic duct lymph were found in follicles. However, the capacity of the bone marrow B cells to enter follicles was very limited. These results are interpreted in relation to previous observations concerning (a) the timing of virgin B cell recruitment into T cell-dependent antibody responses, and (b) the role of B cells in antigen presentation.     

Expression of ICAM-1, VCAM-1 and ELAM-1 in angiofollicular lymph node hyperplasia (Castleman''s disease): evidence for dysplasia of follicular dendritic reticulum cells

The inducible adhesion molecules mediate important functions in the lymphoid tissues. We have investigated the expression of intercellular adhesion molecule 1 (ICAM-1), endothelial leucocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1), and platelet endothelial cell adhesion molecule (PECAM/CD31), using immunocytochemistry on cryostat sections of five lymph nodes from patients with Castleman's disease of the hyaline-vascular type. All five cases were characterized by marked hyperplasia of follicular dendritic reticulum cells, which were extensively present even in the mantle zone. Hyperplastic follicular dendritic reticulum cells showed marked expression of VCAM-1, and weak expression of ICAM-1. In two cases, several dysplastic giant cells with aberrant, polyploid nuclei showed aberrant expression of ELAM-1, an endothelium-restricted molecule. Dysplastic giant cells were positive with DRC-1 (an antibody to dendritic reticulum cells), VCAM-1 and occasionally ICAM-1, were negative for the endothelial cell markers factor VIII-related antigen and CD31 and were non-proliferating (Kl-67-). Cells positive for ICAM-1 or VCAM-1 were rare in the interfollicular areas. In all cases vascular hyperplasia was prominent, but endothelial cells were poorly activated in terms of expression of inducible adhesion molecules and of HLA-DR antigens. The possibility that dysplastic follicular dendritic reticulum cells have a pathogenetic role in Castleman's disease is discussed.     

NEOVASCULARIZATION OF PERICELLULAR FIBROSIS IN ALCOHOLIC LIVER DISEASE

The relationship between sinusoidal capillarization and pericellular fibrosis was studied in 29 specimens of human liver from patients with alcoholic liver disease. Immunohlstochemically, factor VIH-related antigen was not observed in the normal sinusoidal lining cells, but was localized in the capillaries which proliferated in the pericellular fibrotic region. Fibronectln was localized in the proliferated endoplasmic reticulum of newly formed vascular endothelial cells and hepatocytes with the development of pericellular fibrosis, laminin became apparant in the plasmalemmal vesicles of capillary endothelial cells and pericytes and in the endoplasmic reticulum of mesenchymal cells in Disse's space. From these results, It was revealed that pericellular fibrosis was closely related to sinusoidal capillarization. Furthermore, it is suggested that this sinusoidal capillarization may be caused by neovascularization, and that the extracellular matrix produced by endothelial cells, hepatocytes and mesenchymal cells may promote this process.     

Acid cysteine-proteinase inhibitor, a new characteristic of reticulum cells in human lymphoid secondary follicles

Reactive lymph nodes, palatine tonsils and thyroid tissues infiltrated with reactive lymphatic tissue were studied for the presence of immunoreactive acid cysteine-proteinase inhibitor (ACPI). A positive reaction for ACPI was found in germinal centres in all the above tissue types. The morphology and distribution of the positive cells indicated immunoreactivity in the dendritic reticulum cells (DRC). A positive reaction was also found in starry sky cells when a strong starry sky reaction was present in germinal centres. ACPI-positive cells of dendritic appearance were also found outside the secondary follicles in tonsillar tissue from cases of chronic or recurrent tonsillitis, especially between the follicles and the crypt epithelium, in some lymph nodes exhibiting a strong follicular reactive hyperplasia and in thyroid tissue in one case of Hashimoto's thyroiditis. A close anatomical and morphological relationship is pointed out between ACPI reactive dendritic cells and epithelial tissue. This is especially true of the tonsillar crypt epithelium and adjacent lymphoid secondary follicles. The tonsillar crypt epithelium sometimes formed a loose, web-like structure with enmeshed lymphoid cells, even reminiscent of dendritic compartmentalization of lymphoid secondary follicles. The results suggest that ACPI-immunoreactivity of DRC could be a function of the outer membrane of dendritic processes, and thus could be in some way a parallel phenomenon to the morphological expression of the well-established function of DRC in capturing antigen and immune complexes.     

The distribution of immunoglobulin and other plasma proteins in human reactive lymph nodes

The distribution of immunoglobulin, transferrin and albumin in human reactive lymph nodes was determined by the unlabelled antibody peroxidase-antiperoxidase (PAP) method and correlated with the structure of the nodes. All the nodes contained secondary lymphoid follicles which showed polarity towards a lymph sinus, usually the marginal sinus. The lymphatic sinuses were usually dilated. Various types of reticulum cells were demonstrated effectively by the metalophil method. Intracellular and extracellular antigen was well shown in paraffin sections of tissues fixed in buffered formaldehyde containing 5 per cent. acetic acid but cryostat sections were superior for surface Ig. The lymphocyte corona of each follicle reacted for surface immunoglobulin (μ, α, δ κ and λ chains). A polyclonal population of immunoglobulin-containing cells (centrocytes, centroblasts and plasma cells) was present in the follicle centre and numerous plasma cells were often found in the interfollicular areas; each of these cells contained one heavy and one light chain. The many other sites that reacted for immunoglobulin contained not only several heavy chains and both light chains but also transferrin and albumin: these sites included lymphocyte-like cells in the interfollicular areas; histiocytic, dendritic and fibroblastic reticulum cells; clusters of lymphocytes associated with dendritic reticulum cells; clusters of lymphocytes associated with dendritic reticulum cells; the cells lining the lymphatic sinuses and the intrasinusoidal reticulum cells; endothelial cells of the high endothelial venules; “extracellular” material in the germinal centres. The lymphatic endothelium and intrasinusoidal reticulum cells differed from the other sites, however, in reacting strongly for α and J chain and very weakly for γ chain. The fact that in reactive lymphoid tissue many cells contain several plasma proteins makes it difficult to draw conclusions about the role that such cells may have in the immune system. The morphological structure and immunohistochemical findings in ractive nodes are contrasted with the findings in lymphomas.     

TUMOR GROWTH OF THE RETICULOENDOTHELIAL SYSTEM

From the author's hitherto studies on the reticuloendothelial system (RES) it was concluded that the RES is not a single cell system of identical origin, morphology and function but is a group of several types of cells of different origin. From this point of view the heterogeneity of tumors of the RES was studied to reveal the following results. Tumors of the "Reticulo-endothel" (Aschoff) reveal pictures of endothelioma, while tumors of histiocytes in connective tissue show findings of fibrohistiocytoma. Histiocytes and reticulum cells of lymph nodes are respectively independent cells, and reticulum cells do not partake in the development of histiocytosis or histiocytic sarcoma. Follicular lymphoma is a neoplastic growth of reticulum cells having desmosomes in lymph follicles, and tumor cells of the majority of reticulum cell sarcoma are similar to the cells forming the lymph node anlage in the early fetal stage (lymphoreticular cell). Ewing's sarcoma is considered to be a kind of angiopericytoma.     

Histo- and immunopathological features of terminal AIDS. An autopsy case of a Japanese man with neurological signs as initial symptoms

An autopsy case of a 37-year-old Japanese man, confirmed as an AIDS patient infected by an undetermined route of transmission, is presented. The initial symptoms of full-blown AIDS in this case were neurological, and the patient died of severe pneumonia 9 months after onset. The main histo- and immunopathological features were a marked depletion of helper-inducer T cells and dendritic reticulum cells in the lymphoid tissues, opportunistic infections, and some neuropathologic changes. Very few cells, possibly macrophages, immunoreactive with a monoclonal antibody (VAK-5) against HIV-gag protein P24 were found in the mediastinal lymph nodes. Numerous pathogens had induced opportunistic infections in many organs: severe and generalized cytomegalovirus infection, Pneumocystis carinii pneumonia, bronchopneumonia (possibly due to Pseudomonas aeruginosa), candidiasis in the tongue and oral cavity, and atypical mycobacteriosis in the pulmonic hilar lymph nodes. Vascular proliferation was found in the perinodal regions of some lymph nodes, but this was not neoplastic vascular proliferation compatible with that of localized Kaposi's sarcoma.     

Development of antibody-forming cells in follicles of lymph nodes of mice after continuous antigenic stimulation.

It was the purpose of the present study to test a hypothesis on the direct differentiation of newly formed memory B cells into antibody-forming cells (AFC) in the follicles of lymph nodes. AFC may develop in follicles when mobile antigen is present at the moment that such memory B cells have completed their differentiation under influence of immune complexes trapped on follicular dendritic cells (FDC). In order to study whether the simultaneous presence of mobile antigen and immobilized immune complexes on FDC alters the normal distribution of AFC in lymph nodes, different immunization protocols with the thymus-dependent antigens trinitrophenylated keyhole limpet haemocyanin (TNP-KLH) and horseradish peroxidase (HRP) were used. Results confirm that, provided that mobile antigen and immobilized immune complexes are present simultaneously, AFC that are normally found in the medulla of the lymph node may also develop in the follicles.     

AN ELECTRON MICROSCOPIC STUDY ON THE RETICULOENDOTHELIAL CELLS IN THE LYMPH NODES

In an attempt to clarify the cytological characteristics of the RES cells in the lymph nodes and their embryological correlations, lymph nodes and lymph node anlages of germ-free rats, nude mice, and human fetuses were light and electron microscopically examined. On the basis of differences of intracellular organelles, their behaviors for reticulum fibers and of endogenous peroxidatic activity, histiocytes should be reasonably distinguished from the cells conventionally called reticulum cells. Reticulum cells and histiocytes respectively are destined to differentiate in different directions from the early stage of development of the lymph node anlage. Sinus endothelial cells are ontogenetically originated and differentiated from the endothelial lining cells of lymphatic vessels. Primitive reticular cells are differentiated into mature reticulum cells in the lymph nodes, they transform into the lympho-reticular cells, further into lymphoblasts, and finally develop into medium-sized lymphocytes.     

AN AUTOPSY CASE OF PRIMARY ANGIOSARCOMA OF THE PERICARDIUM MIMICKING MALIGNANT MESOTHELIOMA

The pathology of a rare case of primary diffuse angiosarcoma of the pericardium is reported. Grossly, the heart was entirely encased by the pericardial tumor, and the myocardium was only superficially invaded by the tumor. The tumor tissue extended directly to the mediastinum, where the great vessels were embedded in the tumor. A few minute distant metastases were found only in the bilateral lungs and pulmonary hilar lymph nodes. Microscopically, the tumor tissue was composed of malignant cells forming vascular channels admixed with solid areas. Histo- and immunohistochemically, no mesothelial characteristics were evident. Factor VHI-related antigen and Ulex'europaeus I lectin were positive, implying that the tumor was of vascular origin. Grossly, and in part microscopically, this case resembled malignant diffuse mesothelioma, indicating that pericardial angiosarcoma may sometimes mimick malignant mesothelioma. ACTA PATHOL JPN 38: 1345-1351, 1988.     

Localization of antibody-forming cells in draining lymphoid organs during long-term maintenance of the antibody response

Previous studies on the "spontaneous antibody response" have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody-forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.     

Characterization of antibody responses of local lymph nodes to antigen given under the oral submucosa.

We studied the function of submandibular lymph nodes (MLN) in the oral mucosa immune system as compared with that of inguinal lymph nodes (ILN) in the cutaneous one. Primary IgM, IgG and IgA antibody responses in MLN to sheep red blood cells (SRBC) as a model antigen given submucosally occurred more extensively than those in ILN to the antigen injected subcutaneously. Particularly, definite IgA synthesis was seen in MLN but not in ILN. This IgA synthesis was shown to be originated locally in oral submucosal lymphoid tissue or MLN but not in gut-associated lymphoid tissue (GALT). This suggested that the oral mucosal tissue including MLN acts like Peyer's patches in GALT for IgA synthesis. When mice were administered with SRBC and bacterial lipopolysaccharide (LPS) submucosally, the adjuvant effect of LPS was only observed on the capacity of MLN cells for secondary antibody response in vitro. This contrasted to the marked augmentation by LPS of both the primary antibody response in ILN and capacity for in vitro secondary antibody response of ILN cells of mice given SRBC and LPS subcutaneously. The radioactivities detected in the local lymph nodes and other tissues of mice given 51Cr labeled SRBC submucosally or subcutaneously were comparable with each other. MLN, however, contained more Ig+/B220+ B cells and less Thy1+/Ly-1+ T cells than ILN did, and the L3T4/Ly-2 ratio of T cell subpopulations in MLN was lower than that in ILN. Partially corresponding to this observation, the B cell-dependent area was developed more extensively in MLN than in ILN. This difference in cellular composition and organization might in part explain the reason why MLN and ILN display distinct modes of response and sensitivity to the action of LPS.