Rodlet cells in the thymus of the zebrafish Danio rerio (Hamilton, 1822).

The conspicuous rodlet cell (RC) of teleost fishes is a remarkable but still poorly understood phenomenon in the biology of this group. Since their discovery by Thelohan in 1892, a confusing discussion about their nature and physiological function has ensued with various interpretations of RCs as protozoan parasites, gland cells, or a specific type of immune cell. Here we present a study on RCs in the zebrafish Danio rerio (Hamilton, 1822) and the first observation of their occurrence in the thymus, based on light- and electron-microscopy. RCs were confirmed in thymus-surrounding connective tissue, the thymic epithelium and the parenchyma. Very few (late) immature stages of RCs could be identified, already displaying some rodlets and a developing fibrillar cell border but often also signs of a degenerating Golgi apparatus. Apoptotic RCs occurred mostly below the surface epithelium. The mature RCs ejected their rodlets not only at the surface of the epithelium but also into the extracellular environment of the thymic parenchyma. Coalescence of the rodlets into "multi-core" units seems to represent an organ-specific feature. No cellular immune reaction of reticulum cells or granulocytes against RCs and their cellular products was observed. RCs in tight contact with lymphocytes showed conspicuous formation of dense structures in the capsule-like cell membrane, but dense membrane appositions were also observed between RCs and erythrocytes in blood vessels. The possibility of immigration and defective development of RCs in the thymus is discussed.

Platelet-stimulating effects of oxidized LDL are not attributable to toxic properties of the lipoproteins.

One prominent feature of oxidized LDL (OxLDL) is their ability to activate human platelets and effects of OxLDL on platelet function have been shown to depend on the chemical mechanisms that form the basis for the oxidation process. In this regard, the possibility that the observed platelet-stimulating properties of OxLDL might be a direct consequence of cytotoxic effects mediated by these lipoproteins merits further investigation, as experimental strategies to overcome the toxic effects of OxLDL towards a variety of different cell types did not yield conclusive results. In the present work, we show that copper-oxidized LDL mediate severe toxic effects towards a macrophage cell line (decrease in both the number of adherent cells and the amount of incorporated tritiated thymidine, induction of apoptosis and subsequent loss of membrane integrity)--effects that are presumably attributable to products emerging from lipid peroxidation. When added to resting human platelets, copper oxidized LDL stimulate platelets but are not able to trigger an aggregation response on their own. In contrast, hypochlorite-oxidized LDL are able to trigger platelet aggregation, but do not mediate toxic effects towards nucleated cells. Even in the absence of exogenous antioxidants, these lipoproteins mediate cytostatic effects but do not negatively affect cell viability. As a conclusion, platelet-activating effects of oxidatively modified LDL are not attributable to toxic properties of the lipoproteins and this finding might expand possibilities for therapeutical intervention.

Development of the endoplasmic reticulum in the rodlet cell of two teleost species.

The rodlet cell found in different tissues and the blood of teleosts is distinguished by a thick capsule and bipartite rodlets, each consisting of club-like sac and a dense protein core. The development of its rough endoplasmic reticulum (RER) was ultrastructurally investigated in gill and intestinal epithelium of trout (Salmo trutta L., Oncorhynchus kisutch). The RER showed signs of hypertrophy beginning already in immature cells. The typical vesicular appearance noted in the mature cell as well as the apical labyrinth, an accumulation of dislodged mitochondria and vesicles, derives from RER dilatations shedding their ribosomes and pervading the cytoplasm. These dilatations extend also into extracapsular protrusions formerly supposed to be nutritive. A possible role of RER in the formation of the rodlets is indicated by the close association of RER-derived vesicles with rodlet sacs. Conspicuous undulations of the membranes of the sacs as well as the occurrence of tubules (ø = 30 nm) in the dilated RER, rodlet sacs, and along the core support the proposed hypothesis of hypertrophy. Both features signal the storage of (membrane) protein during altered conditions ranging from slowed metabolism to viral infections. The differentiation of apical microvillar projections replaced later by cytoplasmic blebbing and disruption is compatible with surplus production resulting in discharge by pressure. Together with the occasional presence of junctional complexes, these observations argue for an aberrant cell differentiation due to an unknown cause, representing an alternative to the current interpretation of the rodlet cell as a migrating secretory or leucocytic cell with a defensive function.

Membrane transformations in degenerating rodlet cells in fishes of two teleostean families (Salmonidae, Cyprinidae).

Rodlet cells (RCs) of teleosts are identified by their fibrillar capsule and peculiar inclusions, the rodlets, consisting of a club-like sac and a central dense core. Former ultrastructural studies showing signs of hypertrophy of endoplasmic reticulum (ER) were followed up in Salmonids (Oncorhynchus mykiss, Salmo trutta L.) and compared with Cyprinids (Cyprinus carpio L., Carassius auratus L., Alburnus alburnus). Focusing on membrane transformations, unusual undulations of the membranes of rodlet sacs and often apposed ER-membranes, which were observed in mature or discharging cells, increased continuously in degenerating stages and ejected cytoplasmic packages or rodlets. Tubular elements (ø 25-30 nm or 30-50 nm) or small vesicles appeared partly derived from them. Terminal stages of this development were represented by RCs retained in the epithelium, which were completely filled by stacks of tubules and cores. Convoluted membranes were also found persisting between mostly undissolved rodlets at the epithelial surfaces. In Cyprinid species, the membrane changes were less conspicuous but essentially similar, including stages with confluent ER reported only in trout up to now. The membrane transformations resemble structures known as "crystalloid ER" indicating a disturbance in the protein production. The positive immunocytochemical reaction for calreticulin in the rodlet sacs, a luminal ER chaperone mediating recycling of misfolded proteins and upregulated during stress, supports this interpretation. The ER stress-reaction is an evolutionary conservative cytoprotective mechanism during physiological, environmental, and genetic aberrations and fits the increase of RCs reported in quite different situations, although details of its triggering need further investigation.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Direct stimulation of T lymphocytes by immunosomes: virus-like particles decorated with T cell receptor/CD3 ligands plus costimulatory molecules.

Many infectious viruses coevolved with the vertebrate immune system. During the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, called lipid rafts, frequently function as a natural meeting point for viral proteins. The role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and T cell signaling is widely recognized. In our studies, we determined whether lipid rafts, virus budding, and molecular interactions during T cell activation could be brought into a novel context to create artificial antigen-presenting particles. We show here that cell-free virus-like particles (VLP) expressing a surrogate TCR/CD3 ligand (OKT3scFv) and the costimulator CD80 polyclonally activate human T cells independently of accessory cells. VLP expressing the glycoprotein epitope 33-41 of the lymphocytic choriomeningitis virus in the context of H-2D(b) activate and expand naïve, antigen-specific CD8(+) T lymphocytes and differentiate them into cytotoxic effector cells. Efficient targeting of T cell ligands to lipid rafts and ultimately to VLP is achieved by C-terminal introduction of glycosyl phosphatidyl inositol acceptor sequences, replacing transmembrane and intracellular domains. In this work, basic functions of immunostimulatory molecules meet virus biology and translate into a reductionist antigen-specific T lymphocyte-stimulating vehicle, which we refer to as immunosomes. A large variety of agonistic and antagonistic accessory molecules on genuine antigen-presenting cells may complicate the predictable manipulation of T cells as well as the analysis of selected receptor combinations, making immunosomes potentially useful reagents for such purposes in the future.

Ultrastructure of the granulocytes of the South American lungfish, Lepidosiren paradoxa: Morphogenesis and comparison to other leucocytes.

Ultrastructure and peroxidase cytochemistry of the granulocytes of the lungfish Lepidosiren paradoza were investigated. In peripheral blood and hemopoietic tissue (kidney and spleen) only three granulocytic types were found. After comparison with light microscopic images of Giemsa-stained semithin sections the granulocytes were tentatively designated as eosinophilic I, eosinophilie II, and a basophilic type. The eosinophilc I cell was the most numerous granulocyte and was characterized by granules ranging in diameter from 0.2-1.8 µm. A developmental series was observed, leading from small, peroxidase (PO) positive granules to large ones with PO-negative cores and only peripheral PO-positive reactivity. The central electron-dense cores of the large granules were often eroded. The eosinophlic II cell was less numerous than the eosinophilic I type and had smaller (diameter 0.4-0.9 µm) granules than the eosinophilic I type, some of them elongated and rod like. The basophilic cell was rare and contained large, homogenously dense granules measuring up to 1.2 µm in diameter. Immature stages of the granulocytic types showed vesicular rough endoplasmic reticulum. Nuclear identation and segmentation occurred early in development, in contrast to the relatively late onset of these processes in mamals. Monocytes and macrophages had an irregular or slightly indented nucleus and lacked the specific granules of the granulocytes. Phylogenetic relationships were discussed with special reference to the question of the evolution of only one or possibly of two separate heterophilic/neutrophilic cell lines in fishes. © 1993 Wiley-Liss, Inc.

CD32-mediated platelet aggregation in vitro by anti-thymocyte globulin: implication of therapy-induced in vivo thrombocytopenia.

Induction therapy with polyclonal antithymocyte-globulin (ATG) is widely used in the prophylaxis and treatment of acute cardiac-allograft rejection. Thrombocytopenia, however, is a major side-effect of ATG therapy and its mechanisms are poorly understood. The influence of ATG on platelet aggregation was studied aggregometrically, expression of platelet surface activation antigens CD62P and CD63 was determined by flow cytometry analysis, and electron microscopy was utilized to determine thrombocyte morphology. Treatment of platelets with ATG markedly induced aggregation, whereas OKT3 or anti-IL-2R antibodies did not. Furthermore, platelets incubated with ATG featured an up-regulation of the surface activation markers CD62P and CD63, secretion of platelet-bound sCD40L (CD154) and increased signs of aggregation in electron microscopy analysis. The capacity of ATG to induce platelet aggregation was completely blocked by antibodies against the low-affinity Fc IgG receptor (CD32). Since blocking of CD32 abrogates platelet aggregation, we suggest that CD32 plays a crucial role in ATG-induced thrombocytopenia.