In vivo effects of toxic and detoxified endotoxin alone or in combination with muramyl dipeptide on lymphoid and non-lymphoid cells in the spleen of Meth A sarcoma-bearing mice

In this study we confirmed that combinations of toxic or detoxified endotoxin with muramyl dipeptide (MDP) induced much more necrosis of transplanted Meth A sarcoma in mice than toxic endotoxin alone. Detoxified endotoxin and MDP alone had little antitumor effects. We investigated whether these divergent antitumor effects could be related to histopathological changes in the white pulp of the spleen of Meth A sarcoma-bearing mice. Toxic endotoxin reduced the T:B cell compartment ratio in the splenic white pulp by increasing the size of the B-cell compartment while leaving the size of the T-cell dependent inner PALS unaffected. The number of the T-lymphocytes in this area, however, was reduced. The border of B-lymphocytes in the marginal zone was markedly narrowed and the number of marginal metallophils along the inner border of the marginal sinus was decreased. None of these changes were observed after treatment with detoxified endotoxin or MDP. Addition of MDP to either endotoxin did not change their effects. The histopathological changes in the lymphoid and non-lymphoid compartments of the splenic white pulp are apparently exclusively induced by toxic endotoxin. As the antitumor activity of both toxic and detoxified endotoxin combined with MDP are about equal and more powerful than the activity of toxic endotoxin alone, it is concluded that these antitumor effects cannot be related to changes in the white pulp of the spleen.     

Thymus-dependent lymphoid regions in the spleen of the lizard, Calotes versicolor.

This paper describes the distribution of thymus-derived lymphocytes in the spleen of the lizard, Calotes versicolor. Our studies reveal the existence of red and white pulp in the spleen. The red pulp consists of collagenous fibrous septae containing lymphocytes, erythrocytes and sinuses. The white pulp is an area enclosed by fibrous septae, containing lymphocytes, reticular cells and arterioles. The region around the arteriole in the white pulp was depleted of lymphocytes either one month after adult thymectomy or after anti-thymocyte serum treatment. The repopulation of this region was observed in sham-thymectomized controls, but not in thymectomized lizards. Therefore, this peri-arteriolar region may be designated as a thymus-dependent area, as described previously in higher vertebrates. The significance of these findings is discussed in relation to the phylogeny of the immune system.     

The three-dimensional structure of human splenic white pulp compartments.

The precise arrangement of B- and T-lymphocytes in the different compartments of the human splenic white pulp is still largely unknown. We therefore performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The results indicate that the T-cell regions of human spleens may be interrupted by B-cell follicles. Therefore, there is no continuous periarteriolar lymphatic T-cell sheath (PALS) around white pulp arterioles. An arteriole may be surrounded by T-lymphocytes at one level, then run across a follicle without any T-cells around, and finally re-enter a T-cell region. T- and B-cell compartments are intricately interdigitated in the human splenic white pulp. CD4(+) T-lymphocytes and the typical fibroblasts of the T-cell region may extend as a thin shell at the follicular surface within the marginal zone. On the other hand, IgD(++) B-cells continue from the follicular outer marginal zone along the surface of the T-cell region. Our findings indicate that the microanatomy of the splenic white pulp differs between humans and rodents. This may have consequences for the immigration of recirculating lymphocytes and for initial interactions among antigen-specific T- and B-lymphocytes.     

Recovery processes in rabbit spleen after hydrocortisone administration

The effect of hydrocortisone on the rabbit spleen and the recovery processes after the cessation of the hormone injection was studied. A decrease of the absorption function of RES, and spleen atrophy, expressed in a decrease of the organ weight and size followed prolonged hydrocortisons use. Most of the spleen nodes were reduced to an irregular accumulation of lymphocytes. The pyroninophilic cells disappeared in the white pulp. Cessation of the hormone effect was followed by an improvement in the general condition of the animal; the development of the spleen atrophic processes was suspended, the spleen nodes were restored with an increase of pyroninophilic cell count in them. The use of a stimulant under these conditions accelerated the recovery of the animal weight, as well as that of the atrophic spleen, with a normalization of the organ structure.     

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

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

The spleen of the African lungfish Protopterus annectens: freshwater and aestivation

We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6?months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.     

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.     

Morphometric study of the spleen in C57BL strain mice during pregnancy with syngeneic or allogeneic embryos

A morphological study of the spleen in the C57BL line female mice on the 16th day of pregnancy showed a significant increase in the relative area of generative centres and red pulp and a decrease in the white pulp relative area as well as a rise in the amount of plasmatic cells and blast forms in red pulp during allogenic pregnancy (BALB embryos). Such changes are supposed to favour both the preservation of the intact mother-extrafetal organs-fetus system and the improvement of allogenic embryos trophics.     

Immune proteasomes in the developing rat spleen

Changes in the structure of the rat spleen and the distribution of immune proteasomes in it during early postnatal development have been studied using double immunofluorescent staining of tissue sections with antibodies to the LMP7 immune proteasome subunit and to specific markers of T and B lymphocytes. It has been shown that the white pulp on postnatal day 5 is not yet colonized by lymphocytes and contains a smaller amount of immune proteasomes than the red pulp. At this stage, T and B lymphocytes concentrate mainly in the red pulp. On day 8, B lymphocytes occupy the marginal zone, while T lymphocytes aggregate into dense strands close to the white pulp. By day 18, T lymphocytes form periarteriolar sheaths in the white pulp, and the contents of immune proteasomes in the red and white pulp become equally high. An increase in the total content of immune proteasomes in the spleen on the third postnatal week was revealed in our previous study by Western blotting. In addition to T and B lymphocytes, immune proteasomes have also been revealed in other spleen cell types, probably in macrophages and reticular cells of the white pulp. Thus, the postnatal development of the spleen is associated with an increase in the contents of immune proteasomes in it.     

Age and changes in the splenic lymphoid tissue of germ-free rats during the postnatal period of ontogenesis]

In germfree rats the splenic lymphopoiesis is not demonstrated by the investigation to be suppressed, and lymphoid follicules undergo age changes. Up to 15 days, periarterial zone of T-lymphocytes, responsible for cell immunity, is determined, and by the 30th day, as in control animals, peripheral zone of B-lymphocytes, responsible for hormonal immunity, is distinctly observed. In 4-month-old germfree animals, a large amount of free iron crystals is detected in the spleen, while in the organ of control animals its amount is still small. In 10-month-old germfree rats, with the appearance of reactive centers, the zone of B-lymphocytes widens in lymphoid follicules and iron crystals integrate in the red pulp. Lymphopoiesis in the spleen of the germfree rats seems to be maintained by certain local conditions which are connected with increased haemolytic function of the organ. This produces a discharge of a large amount of the products of erythrocytosis which, like autoantigens, stimulate lymphopoiesis in the spleen even when microflora is absent in the organism, while lymphopoiesis in lymph nodes in germfree animals is sharply inhibited.     

Spleen and thymus lymphoid tissue in hypoxia (a biometric study)

The spleen and thymus have been studied macro- and microscopically in rats (180-200 g body mass) on the 1st, 3d, 5th, 7th, 14th and 28th days of adaptation to a decreased atmospheric pressure in the altitude chamber corresponding to lifting to 5,000 and 7,500 m (after a preliminary gradual acclimatization) and on the 14th, 28th, 42d, 56th days of readaptation. A relative mass of the organs, the white pulp section area--the transversal section area of the spleen ratio, the summation section area of its lymph nodules have been estimated. In the thymus the cortico-medullary index (CMI) has been defined. A relative mass of the spleen increases during the first week of hypoxia, and during adaptation period it somewhat decreases and stabilizes, remaining higher than in the control. At the altitude of 5,000 m the cross section area of the lymph nodules decreases by 17% on the 28th adaptation day and at the altitude of 7,500 m--by 27% beginning from the 14th up to the 28th adaptation days. In the thymus the CMI, after some decrease during the first days of hypoxia at the altitude of 5,000 m, increases and normalizes on the 28th adaptation day, and at the altitude of 7,500 m stabilizes on the 14th - 28th days of hypoxia. When the rats are at the altitudes of 5,000 and 7,500 m the thymus lymphoid tissue reacts more quickly to the hypoxia effect and much sooner normalizes during the readaptation period than does the white pulp of the spleen. The main changes in the lymphoid tissue of the spleen and thymus take place on the 7th - 28th days of hypoxia.     

CD8+ T lymphocyte-mediated loss of marginal metallophilic macrophages following infection with Plasmodium chabaudi chabaudi AS

The splenic architecture is essential for the quick resolution of a primary infection with Plasmodium. A critical component of this architecture is the marginal zone (MZ), an area of the spleen that separates the reticuloendothelial red pulp of the spleen from the lymphoid white pulp compartment. There are two unique macrophage populations found in the MZ: MZ macrophages (MZM) found on the outer border of the MZ, and marginal metallophilic macrophages (MMM) found on the inner border, adjacent to the white pulp. We investigated the homeostasis of MMM and MZM following infection with Plasmodium chabaudi and demonstrated that a complete loss of both MMM and MZM occurred by the time of peak parasitemia, 8 days after infection. The loss was not induced by up-regulation of the inflammatory cytokines TNF or IFN-gamma. In contrast, following only CD8+ T cell depletion (not dendritic cell), MMM but not MZM were retained, implicating CD8+ T cells in the P. chabaudi-induced loss of MMM. Retention of MMM occurred in mice deficient in CD95, CD95-ligand, and perforin, indicating that these signals are involved in the death pathway of MMM. These data have significant implications for the understanding of the immune-mediated pathology of the spleen as a result of infection with Plasmodium.     

"Intermediate zone" of mammalian spleens: light and electron microscopic study of three primitive mammalian species (platypus, shrew, and mole) with special reference to intrasplenic arteriovenous communication

The intermediate zone (IZ) of nonperfused and perfused spleens in three species of primitive mammals (shrew, mole, platypus) was studied morphologically. The IZ is a tissue zone consisting of plexiform vessels, probably venous capillaries, and is located transitionally between the white and red pulp. The IZ is separated from the white pulp by the arterial net (AN), in which the white pulp arteries terminate. Development of the IZ differs between the three species examined being distinctive in the platypus and shrew. The IZ is thin in the mole spleen. A closed type of arteriovenous (A-V) anastomosis was demonstrated in or around the IZ in the two Insectivora species examined. In the shrew spleen, peripheral arterial branches running within the IZ anastomose with the AN around the follicle. The AN anastomoses eventually with venous plexiform vessels of the IZ around the nonfollicular area of the white pulp to form a closed system. In the mole spleen, A-V anastomoses were noted between white pulp arteries (follicular and AN) and veins of the red pulp, either by direct communication or through fenestrated IZ vessels compatible with the plexiform vessels of the shrew spleen. A-V anastomosis in the IZ is probable, but not confirmed, in the platypus spleen, as analysis was limited to a nonperfused specimen. Well-developed ellipsoids were noted around arterial terminals of the IZ in the shrew spleen. Ellipsoids were also noted around all arterial terminals of the mole spleen directed to the red pulp. Most ellipsoids of the mole spleen appeared located within the IZ. No ellipsoids were present around arterial terminals of the IZ in the platypus spleen. Closed circulation was noted in terminals of the pulp artery in spleens of all three species. All pulp arteries of the mole spleen are postellipsoid segments of white pulp (AN and follicle) arteries. No ellipsoids were found around terminals of the pulp artery (penicillar artery) in shrew and platypus spleens. The IZ is probably homologous to the perilymphatic sinusoid (vein) of the lungfish spleen and may be regarded as part of the red pulp. The IZ may be representative of primitive mammalian spleens that have closed circulation. The marginal zone (MZ) of common mammalian spleens is probably a modified IZ by differentiation (remodelling) of the intrasplenic vein. In this process, withdrawal of venous vessels from the IZ occurred, leaving a lymphoreticular zone with open circulation (MZ). The marginal sinus reported in some mammalian spleens is probably a modified AN formed during this process. Possible morphological alterations of the spleen in vertebrate phylogeny are discussed.     

Spleen mass as a measure of immune strength in mammals

• 1 In studies of birds and their pathogens, spleen size has frequently been used to make inferences about immune system strength. However, the use of spleen size in mammals is more complicated because, in addition to having an immune function, the mammalian spleen is also a reservoir for red blood cells.
• 2 To assess the reliability of mammalian spleen mass as an indicator of immune activity, we quantified the white and red pulp mass by histology of spleens from shot red deer Cervus elaphus. We then analysed the relationships among spleen mass, the amounts of white and red pulp, and the deer's body condition relative to faecal counts of the nematode parasite Elaphostrongylus cervi.
• 3 White and red pulp mass were positively correlated so that an increase in spleen mass was a positive function of both components of the spleen. In male deer, which had significantly lower body condition and higher parasite loads than females, parasite counts were negatively correlated with spleen mass, white pulp mass, and red pulp mass.
• 4 Our findings suggest that (i) spleen mass in shot red deer is a reliable measure of white and red pulp content; and (ii) when looking at the red deer life history, which is greatly influenced by sex of the deer, splenic mass and white pulp mass could be used as reflections of immune system strength.
• 5 Future studies of mammalian spleens can contribute to the understanding of evolved strategies of immune response investment in mammals. However, determination of the white and red pulp spleen components using various sampling methods must be made prior to their application.

     

The proliferative response of the spleen in cryosurgery.

Using ³H-thymidine autordiography, we studied the cellular proliferation of the spleen of rats after cryolesions in liver, kidney, spleen, and stomach.In the germinal centers at first, a dissociation develops, followed by a hyperplasia with high labeling indices of the germinal center cells with a maximum between the second and third postoperative day. In the surrounding lymphatic mantle zone of the white pulp, as well as in the marginal zone, an increased labeling index of the cells can be observed between the first and second day. The highest percentages of labeled cells in the red pulp are seen on the fifth postoperative day.These cell kinetic results correspond very well with those after antigenic stimulation, for instance, after intravenous injection of sheep erythrocytes. Therefore, these findings suggest that an immunologic reaction occurs in the spleen after cryolesions on parenchymal organs.     

Distribution of prosaposin in rat lymphatic tissues

Prosaposin (PSAP) is as a trophic factor and an activator protein for sphingolipid hydrolase in lysosomes. We generated a specific antibody to PSAP and examined the spatiotemporal distribution of PSAP-immunoreactive (PSAP-IR) cells in the lymphatic tissues of Wistar rats. Immunoblots of tissue homogenates separated electrophoretically showed a single band for PSAP in brain but two bands in spleen. PSAP-IR cells were distributed in both the red and white pulp of the spleen, in both the cortex and medulla of the thymus and in mesenteric lymph nodes. Many PSAP-IR cells were found in the dome portion of Peyer’s patches and the number of PSAP-IR cells increased with the age of the rat. To identify the PSAP-IR cells, double- and triple-immunostainings were performed with antibodies against PSAP, CD68 and CD1d. The large number of double- and triple-positive cells suggested that antigen-presenting cells contained much PSAP in these lymphatic tissues. Intense expression of PSAP mRNA, examined by in situ hybridisation, was observed in the red pulp and corona of the spleen. In rats, the PSAP gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without the insertion. We examined the expression patterns of the alternative splicing forms of PSAP mRNA in the spleen. The presence of both types of mRNA (Pro+9 and Pro+0) indicated that the spleen contains various types of prosaposin-producing and/or secreting cells. These findings suggest diverse functions for PSAP in the immune system.     

Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats.

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0+/-0.02-fold (P<0.05) and PC2 mRNA was increased 1.80+/-0.01-fold (P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.     

Changes in the activity of the reticulo-endothelial system of rats during an infection with T. lewisi.

Data presented show that during the course of a Trypanosoma lewisi infection in rats there was both an activation of the phagocytic cells of the liver and spleen and an increase in their numbers. There was a marked lymphoid hyperplasia in the white pulp of the spleen with an increase in the number and size of the lymphoid follicles. Degenerative changes occurred in the liver parenchymal cells during the infection, and at certain stages large numgers of mononuclear cells were observed in the vascular sinusoids and other vessels of the liver.     

An ultrastructural study of the spleen of the ranid frog Rana perezi

The spleen of Rana perezi is encapsulated by connective tissue and shows by light microscopy two areas with no obvious border: the white pulp and the red pulp. The white pulp-lymphoid clusters are scattered throughout the organ and contain lymphocytes, reticular cells, and some plasma cells. The red pulp displays two different portions. The predominant region consists of reticular cells, lymphocytes, a variety of other leucocytes, and cells undergoing division. This area possibly performs a haemopoietic function. The smaller portion of the red pulp is characterized by reticular-phagocytic cells and may be haemocaretic in its function. Macrophages and pigmented cells occur in both white and red pulp. The organization of the spleen of R. perezi can be considered as a transitional or intermediate state between the primitive condition seen in certain fishes and amphibians and the more complex organ of ammiotes.     

Immune Effects of Nonylphenol on Offspring of Rats Exposed During Pregnancy

Effects of nonylphenol on immune system of male rats were examined. Dams were treated orally with nonylphenol at doses of 0, 20, 40, 80, or 200 mg/kg, respectively, from pregnant days 14 to 19. The offspring rats were investigated at postnatal day 60. Compared with the control groups, the doses of 80 and 200 mg nonylphenol/kg induced an obvious decrease in the absolute and relative weight of spleen and thymus. In the 200 mg/kg nonylphenol-treated group, the proliferative responses of murine spleen lymphocytes cultured in vitro were suppressed, Cytokine productions of interferon-gamma and interleukin-6 in serum were markedly lower than those in the control group. Histologically, the boundary between splenic red pulp and white pulp was unclear, expansion and congestion appeared in splenic sinus, lymphocytes in spleen and thymus dramatically reduced, and lots of focal necrosis cells were present. The results of this study show that nonylphenol can cross the placenta barrier, and that in utero exposure to 200 mg/kg/day nonylphenol can inhibit immune function in male offspring rats.