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

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

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.     

Phagocytosis and lymphocyte migration: evidence that lymphocyte trapping in the spleen following carbon injection is not due to direct lymphocyte-macrophage adherence.

The localization of intravenously injected labelled syngeneic lymphoid cells was studied in the spleen of mice and compared with the localization of Indian-ink-containing macrophages. To distinguish between Indian-ink-containing lysosomes of macrophages and silver grains formed in the autoradiographs over the radiolabelled cells, the latter grains were stained blue by a colour-coupling process. Labelled cells were injected 2 h after the Indian ink. Two hours after their injection the bulk of the labelled cells in the spleen was already localized in the white pulp. At this time the Indian ink had been ingested by macrophages in the marginal zone and to a somewhat lesser extent in the red pulp. Twenty-four hours after injection of the cells their concentration in the white pulp appeared constant or had decreased markedly, dependent on the source of the injected cells (spleen, lymph nodes or thymus). At this time carbon-containing macrophages were also found in the white pulp although they contained less carbon than macrophages in the marginal zone and red pulp. A positive correlation between labelled cells and carbon-containing macrophages was never seen in any part of the spleen. It is concluded that, if carbon-containing macrophages induce lymphocyte trapping, as has been supposed by other authors, this trapping must be mediated by the macrophages in an indirect way, e.g. by soluble mediator molecules released in the circulation.     

Topographical studies of lymphocyte localization using an intracellular fluorochrome

A procedure for analysing the topographical localization in tissue sections or whole-organ mounts of lymphocytes labelled with an intracellular DNA-binding fluorochrome, Hoechst dye No. 33342, is described. The localization of intravenously injected lymphocytes in spleen, popliteal lymph nodes, and Peyer's patches was followed up to 7 days. In the case of spleen, both B and T lymphocytes initially localised in the marginal zone. Subsequently, B cells appeared to exit via the red pulp, while T cells aggregated around vessels in the white pulp. In Peyer's patches, B and T lymphocytes localized to different lymphoid areas. The advantages and potential applications of this technique are discussed.     

The role of macrophages in the immunoadjuvant action of liposomes: effects of elimination of splenic macrophages on the immune response against intravenously injected liposome-associated albumin antigen.

The primary antibody response to intravenously administered and liposome-associated human serum albumin (HSA) was studied in mice under conditions where no response could be detected against the non-liposome-associated form of the antigen. The positive response against the antigen, entrapped in and/or exposed on the surfaces of liposomes, thus resulted from the adjuvant action of the liposomes. In mice intravenously injected with dichloromethylene diphosphonate (C12MDP) also entrapped in liposomes, all red pulp macrophages, marginal metallophilic macrophages and marginal zone macrophages had disappeared from the spleen 2 days after administration. Twenty-two days after such a treatment red pulp macrophages and marginal metallophilic macrophages had reappeared, but marginal zone macrophages were still absent. In mice injected with liposome-associated HSA at 2 days after treatment with the C12MDP liposomes, anti-HSA responses were severely depressed, but administration of the liposome-associated antigen 22 days after C12MDP liposomes elicited a normal response. These results point to a role of splenic macrophages in the processing of liposome-associated antigens, but marginal zone macrophages, which are located close to the open ends of the white pulp capillaries and thus are the first macrophages to meet the antigens arriving in the marginal zone are not required.     

Human spleen microanatomy: why mice do not suffice

The microanatomical structure of the spleen has been primarily described in mice and rats. This leads to terminological problems with respect to humans and their species‐specific splenic microstructure. In mice, rats and humans the spleen consists of the white pulp embedded in the red pulp. In the white pulp, T and B lymphocytes form accumulations, the periarteriolar lymphatic sheaths and the follicles, located around intermediate‐sized arterial vessels, the central arteries. The red pulp is a reticular connective tissue containing all types of blood cells. The spleen of mice and rats exhibits an additional well‐delineated B‐cell compartment, the marginal zone, between white and red pulp. This area is, however, absent in human spleen. Human splenic secondary follicles comprise three zones: a germinal centre, a mantle zone and a superficial zone. In humans, arterioles and sheathed capillaries in the red pulp are surrounded by lymphocytes, especially by B cells. Human sheathed capillaries are related to the splenic ellipsoids of most other vertebrates. Such vessels are lacking in rats or mice, which form an evolutionary exception. Capillary sheaths are composed of endothelial cells, pericytes, special stromal sheath cells, macrophages and B lymphocytes. Human spleens most probably host a totally open circulation system, as connections from capillaries to sinuses were not found in the red pulp. Three stromal cell types of different phenotype and location occur in the human white pulp. Splenic white and red pulp structure is reviewed in rats, mice and humans to encourage further investigations on lymphocyte recirculation through the spleen.     

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。     

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

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

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

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

     

Massive destruction of malaria-parasitized red blood cells despite spleen closure

It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-microm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin beta receptor (LTbetaR(-/-)), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice. Moreover, mice deficient in TNF-alpha, tumor necrosis factor receptor I (TNFRI(-/-)), and LTbetaR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-alpha induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.     

Are murine marginal-zone macrophages the splenic white pulp analog of high endothelial venules?

The entry of lymphocytes into the spleen, in contrast to lymph nodes, does not involve high endothelial venule (HEV) interaction. The precise point of entry, as well as the mechanism by which lymphocytes enter the lymphoid areas of the spleen, remains controversial. We examined in detail the effect of two agents, pertussis toxin (PT) and the sulfated polysaccharide fucoidan, on splenic lymphocyte entry and positioning. These have previously been shown to interfere with lymphocyte extravasation across HEV. PT prevents lymphocyte extravasation, but not binding, to HEV, whereas fucoidan prevents binding and thus subsequent extravasation. Studies presented here show that pretreatment of murine lymphocytes with PT does not numerically affect entry into spleen, but profoundly alters lymphocyte positioning within the spleen. When fluorescently labeled, PT-treated lymphocytes are injected intravenously, they initially accumulate in the marginal zone, in apparent association with the layer of marginal zone macrophages (MZM?) which form a shell around the white pulp. They fail to traverse this layer into the white pulp, and subsequently localize in the red pulp. In contrast, untreated cells initially appear in the marginal zone, then continue to migrate into the white pulp after traversing the MZM? layer. The localization of PT-pretreated lymphocytes adjacent to the MZM? layer is disrupted by intravenous administration of fucoidan. Using a flow cytometric assay of aggregation between MZM? and lymphocytes, we confirmed that fucoidan is also able to inhibit this association in vitro, whereas PT has no effect on this interaction. We propose that MZM? in the mouse are the splenic analog of HEV, forming the port of entry of lymphocytes into the white pulp of the spleen.     

Splenic marginal metallophilic macrophages and marginal zone macrophages are the major interferon-alpha/beta producers in mice upon intravenous challenge with herpes simplex virus

The interferon (IFN)-alpha/beta-producing cells (IPCs) are localized predominantly in the spleen, in particular in the marginal zones (MZ), in C57BL/6 mice injected intravenously (i.v.) with UV-inactivated herpes simplex virus (HSV). We defined the phenotype of these murine IPCs using simultaneous immunohistochemical labelling of intracellular IFN-alpha/beta and various surface antigens. We found that the IPCs in the MZ are not dendritic cells because they did not express major histocompatibility complex (MHC) class II and CD11c molecules. Furthermore, they did not express antigenic markers typical for T cells, B cells or red pulp macrophages. In contrast, the majority of IPCs were stained by the anti-sialoadhesin monoclonal antibody (MoAb) SER-4, which is specific for marginal metallophilic macrophages. In addition, a minor part of the IPCs with a more outward localization in the MZ were stained by a MoAb specific for MZ macrophages. We conclude that the massive IFN-alpha/beta production in the MZ of the spleen upon in vivo stimulation by HSV is mainly exerted by marginal metallophilic macrophages and to a lesser extent by MZ macrophages.     

DEC-205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with 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.     

Splenic macrophages: antigen presenting cells for T1-2 antigens

Thymus-independent antigens type 2 (T1-2) such as DNP-Ficoll or DNP-hydroxyethyl starch are selectively retained in mice and rats by a distinct population of macrophages in the marginal zone of the white pulp of the spleen and in the marginal sinus and septa and medulla of lymph nodes. There is suggestive evidence that the antibody response to the haptenic determinants, which is rapid in onset and persists for many weeks, is largely made by a sessile subpopulation of B cells in the spleen marginal zone characterized by the presence of surface IgM but lacking IgD. Splenectomy in adult mice, rats and humans greatly diminishes and delays the antibody response to DNP-Ficoll, but has no such effect on the response to thymus-dependent, or (in mice) to T1-1 immunogens. In humans, if DNP-Ficoll had been administered prior to splenectomy, a normal antibody response was made when the antigen was readministered after splenectomy, indicating that once the responding B cells had been stimulated they were no longer confined to the spleen. Experiments in mice showed that after elimination of B cells by irradiation, responsiveness to T1-2 antigens could be restored by spleen cells isolated as single-cell suspensions provided that the architecture of the spleen remained, but could not be restored if the mice had been splenectomized. This implies that both the spleen B cells and the marginal zone macrophages are required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphocyte compartments in human spleen. An immunohistologic study in normal spleens and uninvolved spleens in Hodgkin''s disease.

A panel of monoclonal antibodies directed against T and B lymphocyte antigens was used to analyze the presence and localization of several lymphocyte subsets in 13 normal human spleens (3 of newborns) and 17 uninvolved spleens of patients with Hodgkin's disease. The distribution of cells in the white pulp corresponded with findings in other secondary lymphoid organs, except for the presence of a marginal zone, a unique compartment localized at the border of white and red pulp. The phenotype of the marginal zone cells indicates that it is likely that the marginal zone contains nonrecirculating as well as recirculating B cells, while T cells (of the T helper type) are also represented. Therefore, the notion that marginal zone cells are nonrecirculating IgM+, IgD- cells, appears to be an oversimplification. No clear differences were observed between spleens of patients with and without Hodgkin's disease.     

Modulation of Lymphocyte Migration to the Murine Spleen after Marginal Zone Macrophage Phagocytosis of Blood-Borne Particulate Material

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocye Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.     

Modulation of lymphocyte migration to the murine spleen after marginal zone macrophage phagocytosis of blood-borne particulate material

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocye Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.     

Spleen deposition of Cryptococcus neoformans capsular glucuronoxylomannan in rodents occurs in red pulp macrophages and not marginal zone macrophages expressing the C-type lectin SIGN-R1.

The fate of microbial polysaccharides in host tissues is an important consideration because these compounds are often immune modulators. Splenic marginal zone macrophages that express the C-type lectin receptor SIGN-R1, take up neutral polysaccharides such as dextran and the capsular polysaccharide of Streptococcus pneumoniae. Given that the major component of Cryptococcus neoformans capsular polysaccharide, glucuronoxylomannan (GXM), localizes in the spleen when injected intravenously, we investigated whether GXM uptake was mediated by splenic macrophages expressing the SIGN-R1 receptor in mice. No significant differences in the amount and location of GXM deposition were detected in the spleens of mice treated with a SIGN-R1 blocking antibody when compared to controls. Similarly, a blocking antibody to Dectin-1, a co-receptor of -SIGN-R1, had no effects on GXM distribution within the spleen. Histological examination of spleens from mice and rats injected with FITC-Dextran and GXM revealed no significant co-localization, with Dextran and GXM being found in marginal and red pulp macrophages, respectively. Hence we conclude that GXM was not deposited in marginal zone macrophages. However, GXM deposition was found in the red pulp. These results indicate that there is a selective localization of these polysaccharides to different receptors such as SIGN-R1 for FITC dextran in marginal zone and a to-be-identified receptor selectively expressed by red pulp macrophages for GXM.     

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).