Histochemistry and electron microscopy of follicle lining reticular cells in the rat spleen

Summary In the rat spleen cells are found staining with aldehyde fuchsin (AF-cells). Most of these cells are localized at the periphery of the follicles, at the inner border of the marginal sinus. They probably develop in situ. Comparable cells occur in other lymphoid organs. They are able to phagocytize, and resemble also histochemically red pulp macrophages. The aldehydefuchsinophilic granules do not stain for mucopolysaccharides. On the ultrastructural level the aldehydefuchsinophilic granules are represented by cytoplasmic bodies with a faintly granulated matrix. Because of their single membrane and the varying positive reaction on acid phosphatase these bodies are considered to be secondary lysosomes or residual bodies. They contain different materials of unknown endogenous origin. In non-immunized animals the AF-cells fail to show the characteristic dendritic protrusions and infoldings of antigen trapping cells.The cells possess some characteristics of reticular cells e.g. association with reticulin, and electron dense patches on the innerside of the cell membrane at the contact areas. They can be classified among the phagocytic reticular cells forming part of the metalophilic cells in the spleen.     

The significance of paravacuolar granules of the thyroid. A histologic, cytologic and ultrastructural study

The presence of the so-called "paravacuolar granules" in thyroid follicular cells has been associated with increased metabolic activity of the gland, regressive changes, degeneration, phagocytic activity and benign papillary hyperplasia. During the course of a review of the intraoperative cytologic preparations and corresponding histologic sections from 73 thyroid cases, the presence of granules within follicular cells was noted in 25 cases (18 adenomatous or colloid goiters, 3 follicular adenomas, 2 papillary carcinomas, 1 follicular carcinoma and in thyroid tissue surrounding a follicular adenoma in 1 case). Histochemical and ultrastructural studies showed the granules to consist of lysosomes containing hemosiderin or lipofuscin pigments. These findings indicate that the presence of paravacuolar granules in thyroid cells is a common nonspecific finding that simply reflects: (1) the erythrophagocytic capability of the follicular epithelial cells, which results in the accumulation of iron within lysosomes, and (2) the accumulation of lipofuscin pigments within lysosomes as a result of degradation of endogenous cellular material.     

Effect of diethylstilbestrol and related compounds on the Ca(2+)-transporting ATPase of sarcoplasmic reticulum.

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.     

In-vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen

In mice marginal metallophils are located at the periphery of the white pulp along the inner border of the marginal sinus. These cells have a weak phagocytic capacity but their function is still unclear. In the present study evidence is given that marginal metallophils migrate from the periphery of the follicle towards the follicle centres after administration of at least 7 micrograms lipopolysaccharide (LPS). This migration is most significant after 24 and 48 h and appears to be a specific effect of LPS. In the follicle centre marginal metallophils take up cell debris and may become tingible body macrophages. The similarity between these two cell types is discussed. The possible effects of several other polyclonal B-cell mitogens on marginal metallophils have also been studied. Dextran sulphate also induces migration of marginal metallophils but this compound triggers a migration and accumulation of these cells at the periphery of the follicles.     

Inhibitory effect of diethylstilbestrol on histamine release by rat mast cells and its relation to the cellular ATP content

The effect of diethylstilbestrol, a synthetic estrogen, on mast cell secretion was investigated. The results showed that 50 microM diethylstilbestrol inhibited histamine release from rat peritoneal mast cells in the presence and absence of glucose, but did not affect 45Ca uptake stimulated by concanavalin A. Diethylstilbestrol also inhibited histamine release induced by compound 48/80, exogenous ATP, or ionophore A23187. Since estradiol benzoate, hexestrol and daidzein were not inhibitory, the inhibitory action of diethylstilbestrol must be independent of its estrogenic activity. The ATP content of mast cells decreased to less than 0.1 nmol/10(6) cells on treatment with 50 microM diethylstilbestrol at 37 degrees C for 15 min. This effect of diethylstilbestrol in decreasing the ATP content of mast cells correlated well with its inhibitory effect on histamine release. Diethylstilbestrol at 50 microM depleted the cells of ATP at 37 degrees C, but not at 0 degrees C, whereas [3H]diethylstilbestrol ( [monoethyl-3H]diethylstilbestrol) binding to rat mast cells was the same at 0 and 37 degrees C. It is concluded that diethylstilbestrol reduced the ATP content of rat mast cells by inhibiting metabolism of the cells, and consequently inhibited degranulation.     

Appearance of phosphatase reticular cells in the spleen parenchyme of normal and tumor-bearing mice]

The splenic mouse parenchyma presents 3 reticular cells types revealed by histo-enzymatic technics: the phagocytic cells show a strong phosphatasic acid activity, the endothelial cells possess a phosphatasic alcalin or adenosine-triphosphatasic reaction, the perithelial cells are 5' nucleotidasic. These different cells are distributed by forming specific topographic structures in the splenic tissue. The phosphatasic alcalin reticular cells seem to be, with their distribution, characteristic elements of mouse spleen. Indeed the modifications in tumoral animal interest chiefly this cell category. In this case, the reticular cells form a deep and large membrane between marginal zone and perivascular lymphoid sheath of the white pulp. These different reticular cells probably react for the defense system of the animal.     

Competitive affinity chromatography of human alpha fetoprotein on immobilized diethylstilbestrol]

Human alpha-fetoprotein (AFP) was isolated by affinity chromatography method on immobilized diethylstilbestrol from butanol extract of abortive material. Elution from the column was performed with 10% aqueous buffered butanol solution, pH 8.6. During one procedure human AFP-preparation containing about 10% of AFP and about 90% of albumin was obtained, with the yield about 60%. The preliminary incubation of extract of the abortive material with estrone raised AFP yield up to 85% with the increase of AFP content in the preparation up to 35%, and preincubation with estriol and estradiol caused the increase of the yield up to 88-92%, and AFP content in the preparation was 50% and 65%, respectively. The preincubation of human AFP with diethylstilbestrol lowers the yield of this protein, which testified to the possible binding of human AFP with free diethylstilbestrol; testosterone, hydrocortisone and desoxycorticosterone caused the increase of the yield of AFP. So the competitive variant of the affinity chromatography on immobilized diethylstilbestrol makes it possible to raise human AFP preparation purity and yield by decreasing the competition between AFP, and not binding free steroid hormones, ad albumin for immobilized diethylstilbestrol.     

Influence of intercellular contacts in epithelial sheets on the capacity of the cell surface for adhesion and phagocytosis of particles]

The influence of intercellular contacts on the ability of the upper cell surface to adsorb and to phagocytose particles was studied in different types of cultured cells of mouse origin. In cultures of the MPTR strain, cells formed firm contacts which remained unbroken during the epithelial sheet migration into the wound. The contact inhibition of phagocytosis was found in these cultures. The phenomenon involved a low phagocytic activity of the sheet cells which made intercellular contacts in all directions, and of high phagocytic activity of marginal cells which had activity moving free edges. Other epithelial cultures, such as explants of normal kidney and hepatoma 60, behaved similarly. Cultured embryo fibroblasts and hepatoma 22a cells did not form firm intercellular contacts and migrated into the wound one by one. In these cultures most cells had high phagocytic activity. It is suggested that the formation of intercellular contacts alters the upper cell surface ability to adhesion and phagocytosis of particles.     

Neonatal diethylstilbestrol treatment alters aflatoxin B1-DNA adduct concentrations in adult rats

Aflatoxin B1-DNA adduct concentrations were measured in the livers of adult Sprague-Dawley CD rats treated on days 2, 4, and 6 postnatally with 1.45 mumols of diethylstilbestrol and in adulthood with phenobarbital, 3-methylcholanthrene, or vehicle prior to treatment with aflatoxin B1. Aflatoxin B1 (1 mg/kg) was injected 5 hr prior to killing the rats. Female rats exposed neonatally to diethylstilbestrol had significantly higher aflatoxin B1-DNA adduct concentrations (three- to sixfold) than adult female rats treated neonatally with propylene glycol. Liver aflatoxin B1-DNA adduct concentrations were slightly higher in control males as compared to adduct concentrations in neonatally diethylstilbestrol-treated males, as compared to adduct concentrations in control females (not significant [NS]). Phenobarbital and 3-methylcholanthrene treatment followed by aflatoxin B1 injection resulted in decreased aflatoxin B1-DNA adduct concentrations in all rats. Our results demonstrate that neonatal exposure to diethylstilbestrol alters the capacity of adult female rats to form and/or dispose of carcinogen-DNA adducts following a single dose of aflatoxin B1 (increased adduct concentration). This alteration may be a consequence of altered imprinting mechanisms with diethylstilbestrol causing developmental modifications early in life. The animals were, however, able to respond to cytochrome P-450 and P-448 inducers as evidenced by decreased aflatoxin B1-DNA adduct concentrations.     

Malignant uterine mesotheliomas in squirrel monkeys following diethylstilbestrol administration

10 adult female squirrel monkeys were implanted subcutaneously with 4 60 mg pellets of diethylstilbestrol (DES); 4 animals were implanted with cholesterol pellets to serve as controls. Animals were killed at 5, 9, 11, and 14 months after insertion of pellets. All animals implanted with DES had evidence of extreme estrogenic stimulation, including marked edema of the labia, heavy vaginal cornification, and enlargement of the uterine corpus. There were several stages of uterine lesions, in general in proportion to the duration of treatment. The less advanced lesions were restricted to hyperplasia and hyperthrophy of the serosal cells. In more advanced lesions the myometrium was infiltrated; in the most advanced cases there was invasion of the endometrium. The 7 most advanced uterine tumors were classified as malignant mesotheliomas. Cholesterol-treated controls did not have any significant lesions. These observations indicate that DES is carcinogenic in the squirrel monkey, as it has been demonstrated to be in other lower mammalian species.     

Effect of indomethacin on selected immune parameters in different age groups of mice.

The authors studied the effect of indomethacin on the phagocytic activity of polymorphonuclear leucocytes (PMNL) and on haemolytic antibody formation (plaques) by lymphoid cells of the spleen in 3-, 6- and 12-month-old mice. In 3- and 12-month-old animals the phagocytic activity of the PMNL was significantly inhibited. Plaque formation was likewise significantly inhibited in 3-month-old mice, but it was significantly raised in 6- and 12-month-old animals.     

Effect of pretreatment of male Syrian golden hamsters with 7,8-benzoflavone and with diethylstilbestrol on P-450 isoenzyme activities and on microsomal diethylstilbestrol metabolism

Combined treatment of male Syrian golden hamsters with the synthetic estrogen diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF) gives rise to a high incidence of hepatocellular carcinomas, whereas no such tumors are formed with DES alone nor with 7,8-BF alone. To determine whether alterations in DES metabolism may account for the observed hepatocarcinogenicity, we have studied the effect of pretreatment with 7,8-BF alone, DES alone and 7,8-BF plus DES on the levels of hepatic P-450 and cytochrome b5, on the activities of various P-450 isoenzymes and on microsomal DES metabolism. Hepatic P-450 content was significantly increased after pretreatment with 7,8-BF and decreased after DES, while combined pretreatment led to levels similar to those in untreated control animals. Hepatic cytochrome b5 was also elevated in 7,8-BF-treated hamsters; DES pretreatment had no effect, and combined pretreatment led to a slight increase. Four different substrates were used to probe P-450 isoenzyme activity. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin-O-deethylase (ECOD), 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) were all elevated after 7,8-BF-pretreatment, while DES led to a decrease in these activities with the exception of AHH, where a transient increase which was observed after 8 and 20 weeks of pretreatment was back to control levels after 32 weeks. Combined pretreatment with 7,8-BF and DES led to an intermediate response (slight increase) with AHH, EROD and PROD, but not with ECOD, where a full induction comparable with that observed after 7,8-BF alone was elicited. In spite of the modulation of enzyme levels and activities observed after the various pretreatments, the metabolism of DES in microsomes from pretreated animals was virtually identical with that from controls. Therefore it is concluded that modulation of hepatic DES metabolism is not the reason for the observed hepatotumorigenicity; instead, it is speculated that 7,8-BF is the carcinogenic agent in this tumor model, and DES may act as a promotor.     

Dextran sulphate enhancement of lipopolysaccharide-induced tumour necrosis factor-α production by murine peritoneal macrophages: Correlation with macrophage blockade

Abstract Proteose peptone-induced murine peritoneal macrophages (Mø) were preincubated with 100–800 μg/ml of dextran sulphate (DS) 500 ( M _r 500 000) or DS1000 ( M _r 1 000 000). After 2–24 h of the preincubation, the Mø were stimulated with 1 μg/ml of lipopolysaccharide (LPS) in vitro for 18 h in DS-free culture medium. The culture supernatants were then collected for TNF assay. The LPS-induced TNF activity of Mø supernatant preincubated with DS500 or DS1000 for 6 h was enhanced by up to about ten-fold compared with those preincubated without DS. This enhancing effect was not observed when Mø were preincubated with 100–800 μg/ml of low molecular weight DS5 ( M _r 5000) or neutral dextran (Dex) 500 ( M _r 500 000). The enhancement of LPS-induced TNF-α production from Mø was observed after 2 or 4 h of incubation with DS1000 or DS500, respectively. The phagocytic activity of Mø was determined in vitro by the ingestion index and phagocytic capacity using Saccharomyces cerevisiae . Treatment with DS500 or DS1000 significantly suppressed the phagocytic activity from 2 h after the incubation, but this suppression was not observed in Mø incubated with DS5 or Dex500. Our experiments indicate that DS500 and DS1000 act directly on Mø and enhance LPS-induced TNF-α production from Mø, and that the enhancement is closely related to the suppression of Mø phagocytic function.     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

Dendritic cells in the rat spleen follicles

Follicles of peripheral lymphoid organs (rat) contain a type of non-lymphoid cell which is capable of arresting antigen-antibody complexes at the cell surface. These so-called dendritic cells can be visualized in immunized rats by staining antigen-antibody complexes with immunohistoperoxidase techniques. The present study concerns a classification of these cells and comparison with known non-lymphoid cell types such as macrophages, marginal metallophils and tingible body macrophages in the rat spleen follicles. Immunoenzyme histochemical and (enzyme) histochemical techniques have been combined in the same tissue sections to correlate the functional capacity of binding immune complexes with morphological characteristics or phagocytic capacity. Dendritic cells show silver affinity but do not demonstrate a characteristic pattern of hydrolytic enzymes or phagocytosis.     

Prolactin sensitivity of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for alpha-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50-100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports reports had suggested, but rather are generally less sensitive to hormonal stimulants.     

Effects of diethylstilbestrol on the cytogenesis of prolactin cells in the pars distalis of the pituitary gland of the mouse

This study was carried out to examine the developmental stage when prolactin cells differentiate in mice and to examine the effects of diethylstilbestrol on the development of prolactin cells in the fetal and neonatal pituitary glands. A small number of immunoreactive prolactin cells appeared first on embryonic day 15 in control (injected with oil) pituitary glands, whereas they did not increase in number until postnatal day 2. In diethylstilbestrol-treated mice (5 mg/kg body weight, 24 h before killing), a small number of immunoreactive prolactin cells were detectable as early as embryonic day 14, but not on day 13. They increased in number on embryonic days 15 and 16, and decreased markedly on days 17 and 18, followed by a rapid increase after birth. This transient reduction in the response to diethylstilbestrol was partially restored by treatment with metyrapone, a specific inhibitor of corticosteroid production. These results suggest that in the mouse: (1) differentiation of prolactin cells occurs between embryonic days 13 and 14, (2) prolactin gene expression is suppressed in the nascent prolactin cells presumably due to the presence of high levels of estrogen-binding protein, alpha-fetoprotein, and (3) prolactin gene expression is also suppressed by elevation of circulating glucocorticoids during the perinatal period. The present results suggest that, in the mouse, at least a proportion of prolactin cells are not derived from growth hormone cells, because the diethylstilbestrol-induced prolactin cells appear earlier than growth hormone gene expression.     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.     

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.     

The effect of copper deficiency on the formation of hemosiderin in sprague-dawley rats

We demonstrated previously that loading iron into ferritin via its own ferroxidase activity resulted in damage to the ferritin while ferritin loaded by ceruloplasmin, a copper-containing ferroxidase, was not damaged and had similar characteristics to native ferritin (Welch et al. (2001) Free Radic Biol Med 31:999–1006). Interestingly, it has been suggested that the formation of hemosiderin, a proposed degradation product of ferritin, is increased in animals deficient in copper. In this study, groups of rats were fed normal diets, copper deficient diets, iron supplemented diets, or copper deficient-iron supplemented diets for 60 days. Rats fed copper-deficient diets had no detectable active serum ceruloplasmin, which indicates that they were functionally copper deficient. There was a significant increase in the amount of iron in isolated hemosiderin fractions from the livers of copper-deficient rats, even more than that found in rats fed only an iron-supplemented diet. Histological analysis showed that copper-deficient rats had iron deposits (which are indicative of hemosiderin) in their hepatocytes and Kupffer cells, whereas rats fed diets sufficient in copper only had iron deposits in their Kupffer cells. Histologic evidence of iron deposition was more pronounced in rats fed diets that were deficient in copper. Additionally, sucrose density-gradient sedimentation profiles of ferritin loaded with iron in vitro via its own ferroxidase activity was found to have similarities to that of the sedimentation profile of the hemosiderin fraction from rat livers. The implications of these data for the possible mechanism of hemosiderin formation are discussed.