Kinetics of the tingible body macrophage response in mouse germinal center development and its depression with age

Although tingible body macrophages (TBM) have been recognized in germinal centers for over 100 years, their role in the germinal center response is not clear. In this study, the kinetics of the TBM response was quantitatively assessed and correlated with the kinetics of germinal center development in young mice. The TBM response in old mice (which have an age-related depression of germinal center development; Szakal et al., 1990) was analyzed for comparison. Young and old immune mice were challenged with human serum albumin and 0, 1, 3, 5, 7, 10, and 14 days later the popliteal and axillary lymph nodes were evaluated. Germinal centers were localized histochemically in alternate serial sections using horseradish peroxidase conjugated peanut agglutinin. TBM numbers were determined per germinal center on adjacent sections by the presence of tingible bodies or histochemically by using the monoclonal antibody Mac-2. Analysis of lymph nodes from young mice showed that TBM numbers decreased with the dissociation of preexisting germinal centers. TBM reappeared 5 days after challenge and the TBM kinetics paralleled the increase in size of de novo germinal centers. In fact, a constant ratio of one TBM to every 350-450 B cells was maintained from day 5 to day 10. In old lymph nodes, TBM were generally absent throughout germinal center development. The lack of TBM prior to germinal center development and their absence in aged mice are inconsistent with the concept that TBM are required for the induction of the germinal center reaction. However, the data are consistent with a role for TBM in regulating the magnitude of the germinal center reaction.     

Morphological and morphometric studies of the splenic antitumor immune response, elicited by liposome-covered soluble p53 kDa antigen, in chemically-induced rat colon cancer

We evaluated the splenic morphometric changes in rats treated with carcinogen to study development of anti-cancer immune response. When liposome-covered soluble 53 kDa antigen (s53) was injected into these rats, significant tumor-suppression was seen and the percentage of tumor-free animals rose from 15.4% in non-vaccinated rats to 53.8%. In the spleens of carcinogen treated rats that did not develop tumors, activity of B lymphocytes increased significantly. This was manifested by the expansion of the germinal centers to 50.9% of the follicular area reflecting depletion of B cells, and the decrease of the mantle layer to 48.9% of the follicles. A similar picture was seen with T lymphocytes: the area of the marginal zone decreased to 55.2% of the T zone of the white pulp, and that of the periarterial lymph sheaths (PALS) to 33.6%. In tumor-bearing rats features of the immune decompensation were seen: the germinal centers increased to 96.5% of the follicular area, and the mantle layer and PALS decreased significantly. Vaccination prevented these effects, especially in tumor-bearing rats: the PALS occupies 30.4% of the white pulp and the marginal zone 56.1%, and the mantle layer occupied 58.1% of the follicular zone. Similar changes were found in vaccinated rats without tumors reflecting the compensatory character of the immune reaction in vaccinated rats. In conclusion, we found that treatment with carcinogen followed by vaccination with the s53-liposomes complex stimulated the activity of the splenic B, and to a lesser degree the T systems.     

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.     

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.     

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.     

Meckel's diverticulum. II. A novel lymphoepithelial organ in the chicken

We have studied the lymphoid development and structure of Meckel's diverticulum (MD). The lymphoid accumulation began about 2 weeks of age. Between 2 and 5 weeks of age the longitudinal folds were filled with lymphoid tissue. The intensive germinal center formation occurred between 5 and 7 weeks of age. Germinal center formation was associated with the presence of secretory cells. The absence of the secretory cells in the germinal centers was followed by germinal center inactivity which was indicated by the lack of lymphoblasts and the high number of tingible body macrophages. The lymphoid tissue of MD seemed to be fully developed by 10 weeks of age and remained lymphoid at least until 21 months of age. Meckel's diverticulum produced large numbers of plasma cells which were comparable to those of the gland of Harder. We may regard MD as the third pouch of the intestine and suggest that it may be a novel lymphoepithelial organ in the chicken.     

Heterogeneity of macrophages evidenced by variability in their glycoconjugates

Staining rat tissues with a battery of 15 lectin-horseradish peroxidase conjugates showed that macrophages contain glycoconjugates possessing terminal alpha, beta-galactose, N-acetylgalactosamine, fucose, and N-acetylneuraminic acid, plus two terminal disaccharides. Dissimilar binding of lectins by different phagocyte populations in the same or different organs evidenced variability in glycoconjugates according to the location of the macrophages. With a group of four lectins, macrophages stained most intensely in lung, next strongest in splenic red pulp and lymph node sinuses, and weakest in skin and liver. Two populations of macrophages were newly recognized in spleen on the basis of content of fucose-rich glycoconjugate. These included a necklace-like band of macrophages at the border between marginal zone and germinal center and distinctive macrophages dispersed throughout the marginal zone. In lymph nodes, phagocytes stained strongly in the germinal centers and weakly in sinuses for glycoconjugate with N-linked oligosaccharides and conversely for glycoconjugate with terminal beta-galactose. Variable lectin binding indicated heterogeneity of thymic macrophages. Lectin cytochemistry offers increased sensitivity for detecting macrophages in tissue sections, provides selective staining that shows the prevalence and distribution of the phagocytes and differentiates macrophages into separate subtypes.     

摄入不同剂量过量碘大鼠脾脏的形态学改变

目的对摄入不同剂量过量碘的大鼠脾脏进行形态学研究,探讨碘过量对大鼠机体免疫功能的影响。方法断乳1个月Wistar大鼠18只,雌雄各半,分为适碘组(NI)、10倍碘组(10HI组)和100倍碘组(100HI组)。给以不同浓度碘化钾水喂养3个月后,处死取脾脏并称重,对脾脏进行光镜下观察,并对脾小结、生发中心、动脉周鞘和边缘区进行体视学定量分析,对脾脏边缘区免疫细胞进行超微结构观察。结果与正常组比较,10HI组和100HI组大鼠脾脏绝对重量和脾脏指数未见明显改变,10HI组大鼠脾脏脾小结、生发中心、动脉周鞘和边缘区体密度未见明显改变,脾脏免疫细胞超微结构未见明显改变。100HI组大鼠脾脏脾小结、生发中心、动脉周鞘和边缘区体密度明显增加,脾脏免疫细胞超微结构出现明显改变,即呈现功能活跃的表现。结论低剂量过量碘摄入对大鼠免疫功能未产生明显影响,高剂量过量碘摄入可引起大鼠免疫功能亢进。     

Localization in the rat spleen of carbon-laden macrophages introduced into the splenic artery: a subpopulation of macrophages entering the white pulp

Heavily carbon-laden (HC) macrophages, largely derived from the red pulp of the donor spleen, were injected into the splenic artery of recipient rats. Immediately after injection, HC macrophages were found only in the marginal sinus and in the splenic cords. With time after injection, they appeared successively at the periphery of the white pulp, in the deeper white pulp, and finally in and near the germinal centers, suggesting migration of HC macrophages from the marginal sinus towards the germinal centers. The number of HC macrophages in and near the germinal centers reached a peak at 12 h. Most of the HC macrophages in the white pulp were spherical or ovoid in shape with a diameter of 7-11 microns in sections, having an eccentric round or oval nucleus often with a distinct nucleolus and a cap-like or horseshoe-like cytoplasm filled with carbon. When immunostained with monoclonal antibodies against rat macrophage subpopulations, more than 90% of HC macrophages in the white pulp were found to be ED1+2-3-. A population of the same type of macrophages, both in morphology and phenotype, were found in the red pulp of the donor spleen. They were different from the major residents, red pulp scavenger macrophages, which were ED1+2+3- and larger in size and irregular in shape. These results suggest the presence of a distinct subpopulation of macrophages which actively migrate into the splenic white pulp including the germinal centers. A discharge of transferred macrophages from the red pulp to the general circulation is also suggested.     

Antigens in immunity: XVI. A light and electron microscope study of antigen localization in the rat spleen*

This paper describes the ultrastructural location of labelled antigens and carbon in the spleens of rats from 4 minutes to 5 days after injection. Particular attention was focused on the sites of deposition 4 minutes after intra-arterial injection of microgram quantities of ¹²⁵I-labelled Salmonella flagellar antigens, crayfish haemocyanin and BSA, using colloidal carbon for comparison. The combination of radioautography with both light and electron microscopy showed the importance of antigen binding by lymphocytes in the marginal zone of the spleen. Macrophage sequestration of antigens was not prominent in the spleen, although it occurred in the liver with the flagellar antigens and haemocyanin.

In the spleen marginal zone, avid antigen-binding cells were found in situ 4 minutes after the injection of labelled haemocyanin. These appear to be the counterpart in vivo of antigen-binding lymphocytes prepared in vitro. Such cells also occurred infrequently after the injection of labelled polymerized flagellin, but were not found with either BSA or carbon.

The apparent movement of flagellar antigen from the marginal zone to the white pulp between 1 and 2 hours after injection was seen to involve lymphocyte-associated antigen. The follicular antigen localization occurring from 1 day onwards after injection was on the dendritic reticular cells of germinal centres, as has been described in lymph nodes after subcutaneous injection.

Carbon particles were rapidly sequestered in macrophages of the spleen and liver, although some particles were found between cells in the marginal zone for as long as 2 hours after injection. By 2 and 5 days, however, all the carbon was in phagocytes, even in the white pulp. Differences between the localization of antigens and carbon were clear, even in the ultrastructural sites of their location in tingible body macrophages of germinal centres.

The unexpected emphasis of lymphocyte association with labelled antigens in the spleen marginal zone has allowed a revison of the mechanism previously proposed for the movement of antigens within the microenvironments of the spleen.

     

头孢噻甲羧肟对肺炎大鼠免疫应答的调节作用

目的 初步探讨了头孢噻甲羧肟（ＣＡＺ）对肺炎大鼠的免疫调节作用。方法;雄性Ｗｉｓｔａｒ大鼠采用气管内注入感染法制备肺炎克雷伯氏杆菌性肺炎模型,注射治疗剂量ＣＡＺ７ｄ后测定免疫指标：结果ＣＡＺ增强巨噬细胞的吞噬功能及ＡＤＣＣ活性;降低ＣＤ４＾＋Ｔ淋巴细胞亚群,对脾小结发生中心Ｂ细胞密度,ＰＭＮ－ＣＬ及巨噬细胞ＡＣＰ活性无影响,结论：ＣＡＺ可增强肺炎大鼠巨噬细胞和抑制Ｔ淋巴细胞的免疫应答反应。     

Ultrastructural identification of specialized endocytic compartments in macrophages of the thymic cortico-medullary zone and germinal centers of peripheral lymphatic organs of the rat.

In this study transmission electron microscopy was used to investigate the ultrastructural features of macrophages which are strategically positioned in the thymic cortico-medullary zone and the dark zone of germinal centers of peripheral lymphatic organs in adult Wistar rats. We show that this, morphologically distinct, type of macrophage displays the entire range of cytoplasmic inclusions, which structurally closely correspond to those of endosomal/MHC-II-enriched compartments of antigen presenting cells. The macrophages of the cortico-medullary zone and germinal centers contain numerous multivesicular bodies, as well as various kinds of cytoplasmic inclusions ranging from single to aggregated multivesicular/multilamellar bodies to large vacuoles. These multilamellar inclusions are composed of elongated, irregularly shaped cisternae, with abundance of internal membrane profiles and dense bodies. Often, cortico-medullary zone and germinal center macrophages contain the typical multilamellar bodies. Polysaccharides are detected by the thiocarbohydrazide-silver proteinate method within the dense bodies of these macrophages. The functional significance of cortico-medullary zone and germinal center macrophages is briefly discussed.     

Effects of antigen dosage on early localization of specific antibodies in rat splenic germinal centers

In order to identify the initial site of antibody formation in rat spleen, an investigation was made to determine the effects of different antigen dosages on the localization of specific antibodies against sheep erythrocytes (SRBCs). Sixty rats were intravenously injected with 1 ml of either 1%, 5%, or 10% suspensions of SRBCs and killed at days 1, 2, 3, and 4 after immunization. A tissue agglutination procedure in which the binding of SRBCs to cryostat sections of spleen was used to localized anti-SRBC antibodies. Sections used for determination of SRBC binding patterns, and adjacent sections were stained for histological localization or processed for the determination of acid phosphatase (ACP) activity. Spleens of non-immunized rats showing binding of SRBCs closely associated with the ACP-positive marginal metalophils and marginal zone macrophages. This binding was not inhibited by preincubating the sections with 2-mercaptoethanol. The bound SRBCs lysed when incubated with complement. The initial change that occurred after antigen injection was binding over the germinal centers and coronal regions, and heavier binding over the marginal zones that was not associated with ACP-positive cells. In animals immunized with 1% SRBCs, these changes were seen on the third day after immunization. In animals immunized with 1% SRBCs, these changes were seen on the third day after immunization. In animals immunized with 5% or 10% suspensions of SRBCs, these changes occurred 24 hours after immunization, during dissociation of the germinal centers. In later stages there was heavy binding of SRBCs over the white pulp and over the red pulp. Binding induced by immunization was inhibited by pretreating the sections with 2-mercaptoethanol and the bound cells lysed in the presence of complement. The results obtained suggest that IgM antibody to SRBCs appears in the germinal centers at least as early as in the marginal zone or peripheral periarterial region, and support the view that germinal centers may participate in primary antibody responses.     

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).     

THE DEVELOPMENT OF EXTRANODAL LYMPHOID FOLLICLES IN EXPERIMENTAL BRONCHOPNEUMONIA

Extranodal formation of lymphoid follicles was morphologically studied in experimental bronchopneumonia. Control gnotobiotic mice had no peribronchial lymphoid follicles and only lymphatic vessels were traced from the terminal bronchiolar region toward larger bronchi. During the week after intranasal inoculation of mycoplasma pulmonis, lymphoid follicles developed in the terminal portion of the lymphatics by the accumulation of small lymphocytes. A loose network of mesenchymal cells and early infiltration of macrophages, following stromal edema, seemed to play an important role in the early accumulation of lymphocytes. Blastic transformation was seen frequently in the center of the accumulated lymphocytes. Two weeks after inoculation plasma cells emerged conspicuously in the periphery of the lymphoid follicles, and the acute phase of bronchopneumonia began to subside. Typical germinal centers with tingible body macrophages and dendritic reticulum cells developed when the bronchitis perisisted in a chronical manner. ACTA PATH. JAP. 29: 533–543, 1979.     

The development of extranodal lymphoid follicles in experimental bronchopneumonia.

Extranodal formation of lymphoid follicles was morphologically studied in experimental bronchopneumonia. Control gnotobiotic mice had no peribronchial lymphoid follicles and only lymphatic vessels were traced from the terminal bronchiolar region toward larger bronchi. During the week after intranasal inoculation of mycoplasma pulmonis, lymphoid follicles developed in the terminal portion of the lymphatics by the accumulation of small lymphocytes. A loose network of mesenchymal cells and early infiltration of macrophages, following stromal edema, seemed to play an important role in the early accumulation of lymphocytes. Blastic transformation was seen frequently in the center of the accumulated lymphocytes. Two weeks after inoculation plasma cells emerged conspicuously in the periphery of the lymphoid follicles, and the acute phase of bronchopneumonia began to subside. Typical germinal centers with tingible body macrophages and dendritic reticulum cells developed when the bronchitis persisted in a chronical manner.     

Immunohistochemical analysis of the distribution of vimentin in human peripheral lymphoid tissues

The distribution of the intermediate filament protein vimentin in peripheral lymphoid tissues was determined using a monoclonal antibody. Frozen sections of tissue were stained using an avidin-biotin immunoperoxidase method. The antibody stained endothelial cells in spleen, lymph node, and tonsil. Unusual rod-like structures were revealed in the sinusoid-lining cells of the spleen. A variety of reticulum cells was detected, including fibroblastic reticulum cells, histiocytic reticulum cells (tingible body macrophages), and splenic marginal zone macrophages. Very few lymphocytes were immunoreactive. Only weak cytoplasmic immunoreactivity was observed in lymphocytes of the periarteriolar lymphocyte sheath of the spleen. The monoclonal antibody employed appears to be of limited usefulness in detecting normal lymphocytes, but is strongly reactive with endothelial structures and some types of reticulum cells.     

Lymphotoxin-α-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers

Summary: Mice deficient in LTa (LTα-/-) lack lymph nodes and Peyer's patches. This action of LTa in lymph node organogenesis appears to be mediated by the membrane form of LT using a mechanism independent of TNF receptor I (TNFR-I) or II (TNFR-II). In contrast, normal Peyer's patch development appears to require both LTa and TNFR-I, with TNFR-I-/- mice showing hypoplastic Peyer's patch structures. LTα-/- mice also fail to support the normal segregation of T-cell and B-cell zones within the splenic white ptilp. Again, this occurs via a mechanism independent of TNFR-I or TNFR-II. Additionally, follicular dendritic cell (FDC) dusters or germinal centers fail to develop in the spleen of LTα-/- animals. Mice defi-cient in either TNFα or TNFR-I also fail to develop splenic FDC dusters and germinal centers, indicating that signaling by both LTα and TNFα is required for development of these speciahzed lymphoid tissue structures. Finally, the splenic white pulp areas in LTα-/- mice lack the marginal zone of monoclonal antibody MOMA-1-staining metallophilic macrophages, whereas TNFR-I-deficient mice have preserved MOMA-1 staining. Thus, certain actions of LTa to regulate spleen white pulp architecture are medi-ated by receptors other than TNFR-I, most likely by the LTβR or a closely related receptor. We tested whether germinal centers are essential for mat-uration of T-cell-dependent antibody responses. When LTα-/- mice were immunized with low doses of NP-ovalbumin (NP-OVA) adsorbed to alum, there was dramatically impaired production of high afTmity anti-NP IgG; however, after immunization with high doses of NP-OVA adsorbed to alum, LTα-/- mice mounted a high affinity NF-specific serum IgG response similar to wild-type mice, all in the absence of germinal centers or clus-tered FDC. Thus, although germinal centers enhance the processes required for maturation of the humoral immune response, the mecha-nisms responsible for affinity maitiration are not absolutely dependent on the presence of germinal centers.     

Immigration of thoracic duct B lymphocytes into established germinal centers in the rat

Immigration of B lymphocytes into established germinal centers in the rat was studied by transferring genetically marked thoracic duct B cells to non-irradiated congenic hosts at various times between 3 days before and 6 days after host immunization. Seven days after host immunization, the distribution of donor B cells to lymph node germinal centers (relative to their distribution to non-germinal center lymph node areas) was measured by two-color flow cytometry in which (a) donor and host B cells were distinguished by their Ig kappa chain allotypes, and (b) germinal center B cells were distinguished by their lack of labeling with the monoclonal antibody HIS22. Thoracic duct B cells from long-term antigen-primed rats were found to immigrate into host germinal centers much better than B cells from unprimed donors. This effect was antigen specific: primed B cells only immigrated well into host germinal centers induced by the priming antigen. Although B cells localized in germinal centers most efficiently when injected before immunization, specifically primed donor B cells injected after immunization were still found to be at least as evenly distributed to germinal centers as to other lymph node areas, whereas unprimed B cells transferred after immunization localized poorly in host germinal centers. These findings are discussed in light of recent suggestions that memory B cell clones are maintained by continued antigenic stimulation within secondary lymphoid follicles.