Intravenous immunoglobulins induce CD32-mediated platelet aggregation in vitro.

BACKGROUND: Intravenous immunoglobulins (IVIg) and cytomegalovirus immunoglobulins (CMVIg) are currently finding increased acceptance in clinical states of high immune activity and in transplant recipients. A rare side-effect of their application is intravascular thrombosis, which is thought to be related to pre-existing hyperviscosity. In a previous study we have shown that rabbit antithymocyte globulin causes platelet aggregation in vitro via the Fc IgG receptor (CD32). OBJECTIVES: To investigate if IVIg and CMVIg have the potential to cause CD32-dependent platelet aggregation. METHODS: The influence of CMVIg or IVIg on platelets pre-incubated with or without monoclonal antibody AT10 was studied in an aggregometer. Expression of platelet surface activation marker CD62P was determined by fluorescence-activated cell sorting analysis and presence of soluble CD40L (sCD40L) was evaluated by enzyme-linked immunosorbent assay. All in vitro experiments were performed using platelet concentrates from the blood bank, at therapeutic concentrations of immunoglobulins. Results Incubation of platelets with CMVIg and IVIg markedly induced platelet aggregation, and increased expression of CD62P and secretion of sCD40L. The capacity of CMVIg and IVIg to induce platelet aggregation was completely abrogated by adding the blocking antibody AT10 directed against the low-affinity Fc IgG receptor (CD32). CONCLUSIONS: Our results suggest that CMVIg and IVIg solutions with activating Fc domains are able to bind CD32 on platelets and cause platelet aggregation in vitro. These results indicate a mechanism by which in vivo intravascular thrombosis may be explained and suggest caution with concomitant use of packed platelets and IVIg in autoimmune diseases in the clinical setting.

Cytomegalovirus hyperimmunoglobulin: mechanisms in allo-immune response in vitro.

BACKGROUND: Cytomegalovirus hyperimmunoglobulin (CMVIg) containing drugs are routinely administered in cardiac transplantation for prophylaxis against CMV disease. Yet little is known about their influence on transplant relevant immune functions. The aim of this study was to evaluate the effect of CMVIg on cellular immunity in in vitro experiments and to define their role in tolerance inducing mechanisms. MATERIALS AND METHODS/RESULTS: CMVIg reduces proliferation in mixed lymphocyte reactions and anti-CD3 blastogenesis assays and is related to decreased production of immune modulating cytokines interleukin (IL)-2, interferonr (IFNgamma), IL-10. This antiproliferative effect is associated with a cell-cycle arrest in the G0/G1 phase and induction of apoptosis in CD8+ and natural killer cells. Co-incubation with CMVIg causes down-regulation of cell bound immunoglobulin and FcgammaRIII surface expression on natural killer cells and leads to attenuation of antibody dependent cellular cytotoxicity effector functions. CONCLUSIONS: We conclude that CMVIg induces immunological features on leukocytes in vitro that are known to be related to tolerance induction. Our observations extend the current concept of CMVIg as passive CMV prophylaxis to a therapeutic drug compound capable of reducing allogeneic immune response.

Rodlet cells in teleosts: new ultrastructural observations on the distribution of the cores in trout (Oncorhynchus mykiss, Salmo trutta L.).

The distribution of released rodlet cores was ultrastructurally analysed in the epithelia of intestine, kidney tubules and gill of trout (Oncorhynchus mykiss, Salmo trutta L.). In all three tissues, membrane -- bounded cores extruded from the rodlet cells were observed undissolved at the surface of the epithelia, often attached to or between the microvillar borders. Occasionally, in the medium zone of the comparatively high intestinal epithelium, rodlet cells were seen to shed their rodlet sacs into neighbouring intestinal cells. At the epithelial surface the released cores displayed often a conspicuously undulating limiting membrane apparently developing tubular elements (psi 25-30 nm). Additionally, discharged cores were observed in the apical cytoplasm or even in nuclei of damaged or apoptotic cells and in an intraepithelial lymphocyte. In these cases, the cores were often lacking the membrane but showed association with the mentioned tubular elements. In developing rodlet cells the cores originated near or in contact with the nuclear envelope and displayed a polygonal outline and crystalloid substructure. The observed distribution and morphology is unusual assuming the current interpretation of the rodlet cell as defensive, possibly blood-cell derived secretory cell correlated to stress. As crystalline inclusions and undulating, tubulo-reticular endoplasmic reticulum are typical e.g., in excessive protein synthesis, alternative explanations seem worth while considering.

Modification of protein moiety of human low density lipoprotein by hypochlorite generates strong platelet agonist.

Conflicting reports exist about the effects of mildly or extensively oxidized low density lipoproteins (LDLs) on the reactivity of human platelets. This platelet response is mainly caused by modification of the protein and lipid moiety, giving rise to very differently modified species with hardly predictable properties. The aim of this study was to prepare oxidized LDL with modifications essentially restricted to the protein moiety and to determine the eventual platelet responses. We treated LDL at 0 degrees C for 10 minutes with a 60- to 1000-fold molar excess of sodium hypochlorite in borate buffer in the presence of the radical scavenger butylated hydroxytoluene. Under these conditions, neither fragmentation of apolipoprotein B-100 nor formation of LDL aggregates was observed, and lipid oxidation products did not exceed the amount present in untreated LDLs. The degree of modification and the respective effects on platelet function were highly reproducible. Hypochlorite-modified LDLs act as strong platelet agonists, inducing morphological changes, dense granule release, and irreversible platelet aggregation. The evoked platelet effects are completely suppressed by inhibitors of the phosphoinositide cycle but not by EDTA or acetylsalicylic acid. Most likely, these effects are transmitted via high-affinity binding to a single class of sites, which does not recognize native or acetylated LDL. Obviously, modified lysines, and the secondary lipid modifications derived from them, are not essential for this interaction. We conclude that bioactive oxidized lipids are not directly involved in the stimulation of platelets by hypochlorite-modified LDLs.

Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes.

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (M(r)) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in alpha granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

Inhibition of microvascular endothelial apoptosis in tissue explants by serum albumin.

Plasma factors appear to inhibit endothelial cell (EC) apoptosis in vivo so that flow influences microvascular form. The identity of these factors has not, however, been established. Earlier, we reported that apoptosis in isolated, serum-deprived human EC is inhibited by albumin (Alb). Here, we demonstrate likely biological relevance of this to vascular remodelling in experiments with tissue explants. Rat skin explants were incubated in medium M199 with or without serum, bovine Alb, or human Alb. EC in paraffin sections of explants were labelled by lectin histochemistry and the relative proportion of apoptotic was EC determined. Apoptosis was confirmed by transmission electron microscopy and terminal deoxynucleotidyl transferase labelling. Serum-free culture induced EC apoptosis (P < 0.02) and this was strongly inhibited by Alb at physiological concentrations (P < 0.01). This was not a nonspecific protein effect, as mercaptoethanol denaturation destroyed the activity and ovalbumin was not protective. Also, protection was not due to serum contaminants, as recombinant human Alb had activity identical to that of native material. The dose response was identical for all Alb preparations tested, with maximal activity at physiological concentrations. Protection was not limited to rat tissue as similar results were obtained with human gingival explants. These data support a role for Alb as a plasma antiapoptotic factor for EC in tissues.

Depletion of head kidney neutrophils and cells with basophilic granules during peritoneal inflammation in the goldfish, Carassius auratus.

The head kidney morphology of the goldfish with the experimental peritoneal inflammation (on day 2 after i.p. injection with sterile 3% Thioglycollate) is compared with that in the control fish (i.e. on day 2 after i.p. injection with a strile physiological saline PBS) with special emphasis on identification of granulocytes on semithin and consecutive ultrathin sections. The most striking feature of head kidneys of goldfish in the course of peritoneal inflammation is a severe depletion of mature neutrophils and cells with basophilic granules (basophils/mast cells). These observations suggest the involvement of the head kidney, the main hematopoietic organ of teleosts, in the inflammatory process.

Fibrinolytic proteins in apoptotic human umbilical vein endothelial cells.

Endothelial cells express fibrinolytic proteins including: urokinase (u-PA) and tissue type (t-PA) plasminogen activators, type-1 (PAI-1) and 2 (PAI-2) plasminogen activator inhibitors, and u-PA receptor (u-PAR). Apoptotic endothelial cells detach, potentially forming both local and circulating microthrombi in vivo. In this article, apoptotic human umbilical vein endothelium was obtained by serum starvation and compared with nonapoptotic cells rescued from death with fresh medium containing serum. Antigen levels for t-PA, PAI-1, PAI-2, and u-PAR were reduced greatly in apoptosis (p< 0.05). In contrast, u-PA levels were similar in apoptotic as compared with rescued cells (p<0.05). Radioactive amino acids were used to determine absolute levels of protein synthesis and degradation in these cells. Reduced antigen levels likely were due to proteolysis as there was 98% total protein degradation and very little protein synthesis in apoptotic endothelial cells. Also, u-PA levels in apoptotic endothelial cells were not affected by the protein synthesis inhibitor cycloheximide. Endothelial cells in inflammatory sites are exposed to cytokines, which increase both apoptosis and u-PA levels. Data from this article support the idea that maintained u-PA levels in apoptotic endothelium may protect from micro-thrombosis in inflammatory sites as well as in the circulation.

Colocalization of gold-labeled LDL and fibrinogen on platelets: enhanced fibrinogen binding induced by LDL.

Low-density lipoproteins (LDL) specifically bind to the human platelet integrin-alpha IIb and -beta 3 (Koller et al., J. Biol. Chem. 264: 12412-12418, 1989). We show by electron microscopy (EM) that gold (Au)-labeled LDL bind to sites randomly distributed on the surface of platelets in suspension. Within a few minutes, mobile ligand-receptor complexes are translocated from the surface to the open canalicular system (OCS), which finally centralizes as a broad belt. Binding and translocation of Au-LDL are independent of stimulation of platelets by ADP and are completely reversible. Au-fibrinogen shows a strikingly similar, though agonist-dependent, redistribution behavior. Platelets are markedly activated by LDL. This activation is initiated by the binding of LDL to the plasma membrane receptor, and receptor internalization is probably not required for the activation but may instead be one of its consequences. Coincubation with Au-LDL and Au-fibrinogen results in more pronounced activation. The amount of OCS-localized ligands is significantly increased, most likely reflecting enhanced receptor recycling. The two ligands show a tendency to segregate in separate clusters, indicating differences in their postbinding pathways.

Serum albumin is a specific inhibitor of apoptosis in human endothelial cells.

Excess blood vessels are removed by apoptosis of endothelial cells, however, the signals responsible for this have not been defined. Apoptosis of cultured human umbilical vein endothelial cells is induced by deprivation of serum or adhesion. In this paper, apoptosis in human umbilical vein and microvascular endothelium was induced by deprivation of serum and or adhesion. Apoptosis was confirmed on the basis of morphology, ultrastructure and internucleosomal cleavage of DNA. Loss of endothelial adhesion was found to be an early event in cultured endothelial cell apoptosis and was exploited to quantitate apoptosis. The effect of: bovine serum albumin; human serum albumin; recombinant human albumin; dithiothreitol reduced human and bovine albumin; CNBr treated human and bovine albumin as well as ovalbumin upon endothelial apoptosis was determined. Native bovine and human albumin as well as recombinant human material inhibited apoptosis at physiological concentrations with identical dose response curves in both umbilical vein and microvascular cells. Dithiothreitol treatment destroyed all protective activity while bovine but not human albumin was partially inactivated by CNBr treatment. The unrelated protein ovalbumin was not protective. Albumin did not inhibit apoptosis if cells were also deprived of adhesion. The data suggest that albumin is a specific inhibitor of human endothelial apoptosis but does not protect cells also deprived of adhesion. Reduced supply of albumin to endothelium in poorly perfused blood vessels may provide a mechanism for the removal of excess blood vessels in remodelling tissues. Also, the failure of albumin to protect endothelial cells deprived of adhesion from apoptosis may reflect the need to remove potentially micro-embolic cells detached due to trauma.

Ultrastructure of nonspecific cytotoxic cells in teleosts. I. Effector-target cell binding in a marine and a freshwater species (seabream: Sparus aurata L., and carp: Cyprinus carpio L.).

BACKGROUND: Fish cytotoxic effectors form a cell population whose ultrastructure and properties of conjugation with target cells have not been completely established. We report the ultrastructure of the nonspecific cytotoxic cells in a seawater teleost (Sparus aurata L.) and compare it to a freshwater species (Cyprinus carpio L.). METHODS: Blood leucocytes were incubated with HeLa or B16 melanoma cells. Samples were processed for transmission electron microscopic study. RESULTS: Conjugates consisting of leucocytes binding targets were regularly observed after 30 min, 1 hr, or 2 hr of incubation. In both species leucocytes binding to targets showed ultrastructural features of either monocyte-like or lymphocyte-like cells. Monocyte-like cells usually appeared flattened against the targets and seemed to enclose fragments of the target to form cytoplasmic vesicles and the content of their scarce cytoplasmic granules seemed to be delivered into these vesicles. In the seabream lymphocyte-like cells, dense cytoplasmic granules occurred only occasionally, and neither microvilli nor cell processes were present at the contact areas with the targets. In the carp, the contacts were more numerous and formed regularly interdigitating contact areas and the lymphocytes showed granules with characteristic dense and fibrillar contents. CONCLUSIONS: We conclude that seabream and carp have a leucocyte cell population with ultrastructural features of either monocytes or lymphocytes showing nonspecific cytotoxic ability.

Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa.

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

Binding of urinary protein C inhibitor to cultured human epithelial kidney tumor cells (TCL-598). The role of glycosaminoglycans present on the luminal cell surface.

Binding of urinary protein C inhibitor (PCI) to cultured human epithelial kidney tumor cells (TCL-598) was studied. Binding was dose-dependent, time-dependent, and saturable. Heparin interfered in a dose-dependent way with PCI binding to TCL-598 as did heparan sulfate and to a lesser degree also dermatan sulfate. Pretreatment of TCL-598 with protamine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and > 100 micrograms/ml protamine sulfate reduced binding of PCI to < 10% of the control. Binding of 125I-PCI was specific, and bound 125I-PCI was recovered from the cells by heparin treatment or detached together with intact cells by EDTA treatment, migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound 125I-PCI. Furthermore, cell-bound PCI was functionally active as judged from its ability to inhibit the amidolytic activity of urokinase, and its inhibitory activity was stimulated approximately 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the luminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of heparin (50 micrograms/ml) and after pretreatment of the cells either with protamine sulfate (400 micrograms/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cells with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approximately 70% after chondroitinase ABC treatment of the extracellular matrices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximately 95%. These data suggest that heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfate-containing proteoglycans, the overall predominant PCI-binding proteoglycans in TCL extracts, are responsible for PCI binding to the extracellular matrix. Heparan sulfate, however, exposed to an environment containing PCI under physiological conditions, might localize PCI and modulate its target enzyme specificity in vivo.

Ultrastructural analysis of leucocyte interaction with tumour targets in a teleost, Cyprinus carpio L.

The first step in natural killer activity, target cell binding, was investigated ultrastructurally in order to identify presumptive cytotoxic cells. After incubation of unfractioned peripheral blood cells of the carp, Cyprinus carpio L., with HeLa tumour cells as targets, regular conjugate forming was observed. Targets were surrounded by cells bearing features either of lymphocyte or monocyte/macrophage types. Cells with the usual lymphocytic characteristics contained acid phosphatase positive granules of various density and - after two hours of incubation - paracrystalline inclusions were observed. Cells with monocyte/macrophage morphology were characterized by abundant organelles, varying granules and occasional phagolysosomes. In both cell types development of the contact area was similar to those events known from mammalian cytotoxic cells: development of interdigitating microvilli and increase in the reoriented secretory apparatus (both more intense in the monocyte/macrophage cells than in the lymphocytes). The possibility of several cytotoxic cell types instead of one primitive cytotoxic precursor type proposed earlier in literature is discussed.

New aspects on the 'rodlet cell' in teleosts.

The enigmatic rodlet cells, hitherto interpreted mainly as unidentified parasitic stages ('Rhabdospora thélohani') or as a sort of secretory cell, have been studied with electron microscopical and histochemical methods in three teleost species (Cyprinus carpio L., Tinca tinca L., Salmo gairdneri Rich.). The typical inclusions, the rodlets, consist of a dense core and a less dense cortex, with a coarse flocculent outer zone. The latter shows a positive reaction for neutral polysaccharides respectively glycoproteins (with the silver proteinate method and the equivalent silver methenamine-staining), concurring with some of the light microscopic data. While this suggests the secretion of this material by the Golgi apparatus, the core gives contradictory results, pointing to genetic material instead of a secretory product. Although the Feulgen method (as standard test for DNA with only a few exceptions known) is negative, DNA is indicated by several other methods (light microscopical fluorescence staining with HOECHST 33258, EDTA-method after Bernhard (1969), enzyme digestions positive with DNAse and negative with RNAse). Taken together with some observations on the tissue reaction (phagocytosis of the often discharged rodlets by macrophages and also the more specifically acting heterophilic granulocytes; isolation of the rodlet cells from the adjoining tissue as seen e.g. with the ruthenium red method), an exogenous parasitic nature of these cells seems more plausible, even if the evidence is not sufficient for an exact classification of either the cell or its characteristical inclusions.

Developmental stages and localization of peroxidatic activity in the leucocytes of three teleost species (Cyprinus carpio L.; Tinca tinca L.; Salmo gairdneri Richardson).

The ultrastructural localization of peroxidase (PO) in the leucocytes of three teleosts (Cyprinus carpio L., Tinca tinca L., Salmo gairdneri R.) has been investigated using the 3,3'-diaminobenzidine method. In the heterophilic granulocytes the granules show a species specific structure and are PO-positive at pH 7.6. They can be traced back to small granules arising near the Golgi apparatus (GA) in the promyelocyte. They coalesce to form larger granules and gradually change into the mature type. Myelocytes contain small unreactive granules, and these represent a second granule population. Eosinophils contain one PO-positive granule type (at pH 9), and these granules show a varying density during cell maturation. Basophils are present only in the Cyprinid species, and contain unreactive granules originating from precursors displaying a weakly positive reaction at pH 7.6. The active secretory organelles (RER, GA) are PO-negative, except for a weekly positive reaction in the flocculent matrix of the inner G-cisternae. In promonocytes and monocytes the granules are unreactive, but in the macrophages PO-positive staining occurs in a few small to medium sized granules, and in large vacuoles. A least some of these latter are apparently derived from phagolysosomes containing digested erythrocytes. Thrombocytes and lymphocytes are unreactive. The successive development of PO-positive and negative granule populations in the heterophils, and the PO-reactivity of eosinophils and basophils, show some similarities to the corresponding cells in higher vertebrates, but an analogous PO-positive ("azurophil") granule type in monocytes seems to be absent.

Structure and ribonucleoprotein staining of kinetochores of colchicine-treated HeLa cells.

HeLa cells treated with colchicine show a lengthening of kinetochores lacking an inner layer. Combined EDTA- and ribonuclease treatment (BERNHARD 1969) indicate the presence of ribonucleoprotein in the dense outer layer as well as in the surrounding fine fibrillar mass. Reconstructions from serial sections reveal a band-like enlargement of these layers around the primary constriction during the inhibition of mitosis by colchicine.

[New findings on the structure of chromosomes (author's transl)]. / Neue Befunde zur Struktur der Chromosomen

The structural element of eukaryotic chromosomes is the chromatin fibre consisting of histones and DNA. The chromatin fibre is about 100--200 A thick. One chromatid is built up from one chromatin fibre running through from one end to the other and laid in numerous irregular foldings. The chromatin fibre is a chain of nucleosomes. These are globular histone bodies around which the DNA winds. Nucleosomes can be observed in isolated chromatin fibrils as well as in thin sections of chromosomes after different modes of fixation. Prophasic chromosomes or early premature condensed chromosomes are thin uncoiled threads. With chromosome condensation a major coiling is seen. No constant regular arrangement of the chromatin fibril besides the major coils is observed. A rather diffuse decondensation takes place in ana- and telophase.