David N. Skilleter

A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

A comparison of the accumulation of ricin by hepatic parenchymal and non-parenchymal cells and its inhibition of protein synthesis

Rat liver non-parenchymal cells in vivo were found to accumulate 125I-labelled ricin to a much greater extent than parenchymal cells. Similarly, in monolayer cell cultures, the rate of ricin uptake by non-parenchymal Kupffer cells was several times that by parenchymal cells. Evidence is provided also to suggest that ricin is primarily recognized by Kupffer cells via terminal mannose residues in the toxin, whereas ricin uptake by parenchymal cells was consistent with a role of the previously postulated galactosyl-containing cell receptors. Protein synthesis in Kupffer cells in vitro, although observed to occur at a lower rate than in parenchymal cells, was 100--1000-times more sensitive to inhibition by ricin. The selective damage known to be caused to liver sinusoids by ricin, therefore, may reflect both the relative efficiency with which the toxin is taken up by these cells and the extreme sensitivity of protein synthesis in the cells to inhibition by ricin.

Selective uptake of ricin A-chain by hepatic non-parenchymal cells in vitro: Importance of mannose oligosaccharides in the toxin

Free ricin A‐chain was actively taken up in vitro by rat liver non‐parenchymal cells but not by parenchymal cells. A‐chain uptake by non‐parenchymal cells could be selectively inhibited by D‐mannose, L‐fucose or ovalbumin and was markedly decreased after partial removal of mannose residues from the oligosaccharides present in the glycoprotein by enzymic deglycosylation. Uptake of free ricin B‐chain by non‐parenchymal cells was greater than that by parenchymal cells but in both cases was little influenced by enzymic deglycosylation of the glycoprotein. The results are consistent with mannose receptor recognition of ricin A‐chain by non‐parenchymal cells and have important implications for the clinical use in vivo of antibody‐ricin A‐chain conjugates in cancer therapy.

Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non-parenchymal cells of rat liver

The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non-parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non-parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route.

Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.

The uptake and subsequent loss of beryllium by rat liver parenchymal and non-parenchymal cells after the intravenous administration of particulate and soluble forms

Abstract A cell isolation technique has been used to study the uptake and subsequent loss of beryllium (Be) by rat liver after intravenous administration of non-lethal doses of either particulate beryllium phosphate or the more hepatotoxic soluble BeSO4. It has been shown that beryllium phosphate is removed from the blood predominantly by the non-parenchymal (sinusoidal) cells of the liver and to a lesser extent more slowly by the parenchymal cells. After 24 h when the parenchymal cells have reached maximal Be content there has been a 50% loss of Be from the non-parenchymal cells and a similar loss from whole liver which is reflected in an increased level of Be in the blood. The Be count of non-parenchymal cells subsequently decreases much more slowly in a manner similar to that of the parenchymal cells, both being only halved during the following week. Within 24–48 h some redistribution of Be to the spleen occurs and it is suggested that this in part may be the result of Kupffer cell death. In splenectomized animals a high proportion of this redistributed Be appears to be retaken up by the liver mainly by the parenchymal cell population. After administration of BeSO4, which is known to form beryllium phosphate in plasma, a greater proportion of the Be is taken up slowly by the parenchymal cells and no redistribution of Be to the spleen is observed. It is suggested that this behaviour is related primarily to the smaller size and nature of the beryllium phosphate particles formed in plasma under these conditions. The rate of loss of Be from both the parenchymal and non-parenchymal cells is similar to that measured in beryllium phosphate treated animals. It has been estimated that liver cell death is produced when the cell content exceeds 2–3 nmol Be/106 cells although parenchymal cells appear to be more sensitive to Be derived from BeSO4 than preformed beryllium phosphate.

Mitogenic effects of beryllium and zirconium salts on mouse splenocytes in vitro

The beryllium (Be) and zirconium (Zr) salts, BeSO4 and Zr(SO4)2, each exerted a concentration-dependent stimulation of mouse spleen cell proliferation as measured by an increase in [3H]thymidine incorporation into lymphocyte DNA, although the maximal response induced by Zr(SO4)4 (4-5 fold at 100-200 microM) was greater than that by BeSO4 (2-3 fold at 1-5 microM). Preincubation of splenocytes with low concentrations of BeSO4 (less than 1 microM) or a broad range of Zr(SO4)2 concentrations (2-100 microM) was also found to assist subsequent lectin (concanavalin A; ConA)-mediated lymphocyte proliferation. The results indicate that at defined concentrations Be and Zr salts can both act as lymphocyte mitogens and augment the functional responsiveness of immune cells, which may help explain the characteristic induction of delayed hypersensitivity and production of immunological granulomas by these metals in vivo.

Mannose receptor dependent uptake of a ricin a chain - antibody conjugate by rat liver non-parenchymal cells

Mannose receptor mediated uptake by the reticuloendothelial system has been suggested as an explanation for the rapid removal of ricin A chain antibody conjugates from the circulation after their administration. We have measured, in the rat, hepatic uptake of a ricin A chain antibody conjugate in vivo and its susceptibility to inhibition by a mannosylated protein and have measured uptake of the conjugate in vitro by rat parenchymal and non-parenchymal liver cells. The results indicate that rapid hepatic uptake of conjugate does occur in vivo; cultured non-parenchymal cells accumulate the conjugate to a much greater degree than cultured parenchymal cells and that mannose receptors appear to be involved in the process.

Biochemical properties of beryllium potentially relevant to its carcinogenicity

The carcinogenicity of beryllium to several animal species is well established and evidence exists which strongly suggests that this is the case in human exposure. In this review several biochemical properties of the metallocarcinogen are considered including, the causation of cell transformation, and infidelity of DNA synthesis, inhibition of cell division and enzyme induction, and interference with regulatory mechanisms controlling gene expression. These effects are discussed in relation to beryllium chemistry, cellular accumulation mechanisms and distribution to subcellular organdies and molecular targets. It is suggested that the ultimate location and interactions of the metal ion in cell nuclei and its selective inhibition of certain protein phosphorylation reactions in particular are the biochemical effects potentially most relevant to induction of beryllium carcinogenesis.