Fas/CD95/APO-1 can function as a death receptor for neuronal cells in vitro and in vivo and is upregulated following cerebral hypoxic-ischemic injury to the developing rat brain.

Fas/CD95/Apo-1 is a cell surface receptor that transduces apoptotic death signals following activation and has been implicated in triggering apoptosis in infected or damaged cells in disease states. Apoptosis is a major mechanism of neuronal loss following hypoxic-ischemic injury to the developing brain, although the role of Fas in this process has not been studied in detail. In the present study, we have investigated the expression and function of Fas in neuronal cells in vitro and in vivo. Fas was found to be expressed in the 14 day old rat brain, with strongest expression in the cortex, hippocampus and cerebellum. Cross-linking of Fas induced neuronal apoptosis both in neuronal PC12 cells in culture and following intracerebral injection in vivo, indicating that neuronal Fas was functional as a death receptor. This death was shown to be caspase dependent in primary neuronal cultures and was blocked by the selective caspase 8 inhibitor IETD. Finally, cerebral hypoxia-ischemia resulted in a strong lateralised upregulation of Fas in the hippocampus, that peaked six to twelve hours after the insult and was greater on the side of injury. These results suggest that Fas may be involved in neuronal apoptosis following hypoxic-ischemic injury to the developing brain.

CD45 engagement induces L-selectin down-regulation.

The CD45 glycoprotein isoforms exhibit a receptor-like composition and display intracellular protein tyrosine phosphatase (PTPase) activity. The present study links CD45 to the regulation of L-selectin (CD62L), a leucocyte glycoprotein important for extravasation and homotypic aggregation. Monoclonal antibodies (MoAbs) IOL1b and AICD45.2, but not GAP8.3, all of which are directed against common CD45 epitopes, were found to elicit lymphocyte L-selectin down-regulation. Lymphocyte L-selectin down-regulation in response to anti-CD45 MoAbs was enhanced by high cell density and partially antagonized by the protein tyrosine kinase (PTK) inhibitor, herbimycin A. The MoAbs IOL1b, AICD45.2 and GAP8.3 recognized granulocyte-expressed CD45 but did not induce loss of L-selectin expression of granulocytes. In contrast, the CD45 PTPase inhibitor, vanadate, induced L-selectin down-regulation both in lymphocytes and granulocytes. The PTPase activation by nitric oxide (NO) or the NO-generating compound, sodium nitroprusside, did not affect L-selectin surface expression. Increased concentrations of soluble L-selectin were detected after anti-CD45 or vanadate-induced down-regulation of L-selectin surface expression. While activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces rapid L-selectin down-regulation of L-selectin surface expression in both lymphocytes and granulocytes, the PKC inhibitor, H 7, was also found to down-regulate lymphocyte and granulocyte L-selectin surface expression. The inhibitor H 7 synergized with vanadate in down-regulating lymphocyte L-selectin surface expression, but partially inhibited vanadate-induced granulocyte L-selectin down-regulation. The results suggest that in a cell type-specific fashion the PKC system and tyrosine phosphorylation and dephosphorylation cascades are involved in the regulation of L-selectin surface expression.

Cerebrospinal fluid soluble L-selectin (sCD62L) in meningoencephalitis.

The leucocyte adhesion molecule L-selectin (CD62L) is rapidly cleaved off proteolytically after cell activation, generating soluble L-selectin (sCD62L) molecules. sCD62L concentrations were determined in 185 cerebrospinal fluid (CSF) samples obtained from children aged 1 month to 17 years. In 36 CSF samples of children with meningoencephalitis, sCD62L was significantly higher (median 209 fmol/ml) than in samples of children with other febrile diseases (n = 67, median 50 fmol/ml) or non-febrile disorders (n = 82, median 44 fmol/ml). There was a positive correlation between CSF protein and CSF sCD62L (rS = 0.68), suggesting that a disturbed blood-brain barrier contributes to raised sCD62L concentrations in the CSF. However, the CSF sCD62L/protein ratio of children with meningoencephalitis was significantly higher than in children with other febrile diseases or non-febrile disorders, indicating that sCD62L concentrations in children with meningoencephalitis were higher than expected from plasma leakage alone. It is concluded that both an impaired blood-brain barrier and the generation of sCD62L by infiltrating leucocytes contribute to raised CSF sCD62L concentrations in children with meningoencephalitis.

Soluble L-selectin (sCD62L) umbilical cord plasma levels increase with gestational age.

L-Selectin (CD62L) is a leukocyte surface membrane glycoprotein involved in extravasation and homotypic aggregation which is rapidly cleaved off after cellular activation. From culture supernatants and body fluids, soluble L-selectin (sCD62L) has been recovered with its functional activity retained. We devised a sensitive enzyme-linked immunoassay for quantitation of sCD62L which was used to measure sCD62L in umbilical cord plasma of 255 human newborns with a gestational age (GA) of 23-43 wk (median 38 wk). sCD62L levels ranged from 1.14-13.8 pmol/mL (median 7.2 pmol/mL) and showed strong correlations with GA (r = 0.71, p < 0.001), birth weight (r = 0.66, p < 0.001), and absolute neutrophil cell counts (ANC) (r = 0.62, p < 0.001) obtained from a peripheral vein within the first 6 h of life (n = 153), whereas there was a weak inverse correlation with absolute normoblast counts (r = -0.27, p < 0.001). In multiple regression analysis, only GA and ANC retained a significant association with sCD62L levels (p < 0.001). Decreased sCD62L levels were found to be associated with multiple gestation (4.8 +/- 2.4 pmol/mL versus 7.7 +/- 2.3 pmol/mL, p < 0.05) also when considering GA and ANC as covariates. In contrast, increased sCD62L levels in infants born from meconium-stained amniotic fluid, and decreased levels in newborns with acute bacterial infection could be fully attributed to differences in GA and ANC.(ABSTRACT TRUNCATED AT 250 WORDS)

L-selectin is down-regulated in umbilical cord blood granulocytes and monocytes of newborn infants with acute bacterial infection.

The leukocyte glycoprotein L-selectin mediates an early step in the recruitment of leukocytes to sites of inflammation. L-Selectin surface expression is rapidly down-regulated by inflammatory signals in vitro. In a prospective study, we found L-selectin expression on umbilical cord blood granulocytes and monocytes to be significantly decreased in newborn infants with acute bacterial infection compared with controls (p < 0.01). A significantly reduced L-selectin expression of both granulocytes and monocytes was also found to be associated with an increased neutrophil immature/total ratio (p < 0.01) but not with other laboratory markers of neonatal sepsis. There was no apparent impact of prematurity, low birth weight, gestational hypertension, or gestational diabetes on L-selectin expression. Although the mode of delivery did not affect granulocyte L-selectin expression, umbilical cord blood monocytes showed an increased L-selectin expression after emergency cesarean delivery compared with samples obtained after elective cesarean or vaginal delivery (p < 0.01). We conclude that acute systemic inflammation results in down-regulation of granulocyte and monocyte L-selectin expression in vivo similar to that observed in vitro.

Down-regulation of L-selectin surface expression by various leukocyte isolation procedures.

L-selectin, a cell surface glycoprotein expressed on lymphocytes, granulocytes, and monocytes, has been implicated in lymphocyte homing and extravasation of phagocytic leukocytes into areas of inflammation. Considerable differences of L-selectin expression among various individuals has been reported, with clinical correlations to perinatal events, maturation, and circadian rhythm. In this study, L-selectin expression of various white blood cells was found to be differentially sensitive to ficoll-hypaque or percoll density gradient centrifugation. After density gradient centrifugation, a significant loss of median monocyte L-selectin expression was observed when compared to time and temperature-matched controls or results obtained by whole blood incubation with anti-L-selectin monoclonal antibodies followed by simultaneous leukocyte fixation and red cell lysis. Mock treatment itself was associated with a variable L-selectin loss of monocytes but not lymphocytes or granulocytes. Ficoll-hypaque or percoll density gradient centrifugation resulted in significant L-selectin down-regulation of lymphocytes while granulocytes separated from lymphocytes and monocytes by ficoll-hypaque or percoll retained full L-selectin surface reactivity. L-selectin downregulation was seen also after colloid sedimentation with hydroxy-ethyl starch. It is concluded that unseparated blood should be used for measuring L-selectin expression.

Down-regulation of L-selectin surface expression by various leukocyte isolation procedures.

L-selectin, a cell surface glycoprotein expressed on lymphocytes, granulocytes, and monocytes, has been implicated in lymphocyte homing and extravasation of phagocytic leukocytes into areas of inflammation. Considerable differences of L-selectin expression among various individuals has been reported, with clinical correlations to perinatal events, maturation, and circadian rhythm. In this study, L-selectin expression of various white blood cells was found to be differentially sensitive to ficoll-hypaque or percoll density gradient centrifugation. After density gradient centrifugation, a significant loss of median monocyte L-selectin expression was observed when compared to time and temperature-matched controls or results obtained by whole blood incubation with anti-L-selectin monoclonal antibodies followed by simultaneous leukocyte fixation and red cell lysis. Mock treatment itself was associated with a variable L-selectin loss of monocytes but not lymphocytes or granulocytes. Ficoll-hypaque or percoll density gradient centrifugation resulted in significant L-selectin down-regulation of lymphocytes while granulocytes separated from lymphocytes and monocytes by ficoll-hypaque or percoll retained full L-selectin surface reactivity. L-selectin downregulation was seen also after colloid sedimentation with hydroxy-ethyl starch. It is concluded that unseparated blood should be used for measuring L-selectin expression.

Polarization-optical investigation (topo-optical analysis) of the structure of the human erythrocyte glycocalyx. Influence of pH, ionic strength and diamide-induced spectrin cross linking.

The value of membrane anisotropy after fixation and topo-optical analysis of erythrocytes stained with toluidine blue is a measure for the degree of spatial order of the dyestuff-binding acidic residues of the glycocalyx and thus a parameter for the characterization of the glycocalyx structure. Lowering the pH value and/or the ionic strength of the staining medium results in a decrease of membrane anisotropy indicating a lower order of the anionic residues. In agreement with the findings of other authors this phenomenon seems to be connected with an expansion of the glycocalyx. Diamide-induced oxidative crosslinking of spectrin before fixation and staining with toluidine blue at physiological pH and ionic strength also results in a decreased anisotropy. This indirect influence on the glycocalyx structure may be caused by an increase of the charge density within the glycocalyx due to a diamide-induced rearrangement of the membrane skeleton and of the transmembrane proteins bound to it.

[Structure and function of human erythrocyte membrane skeletons]. / Struktur und Funktion des Membranskeletts des menschlichen Erythrozyten.

On the basis of extensive studies of the literature and of own results the present knowledge about the structure of the membrane skeleton of human erythrocytes is summarized and functional and clinical aspects are described. The spectrins are the centre of interest. Their interconnections, spatial arrangement and association with other components of the membrane are explained in greater detail. With regard to the membrane skeleton questions of erythrocyte shape, membrane integrity, phospholipid asymmetry, distribution of transmembrane proteins and cell deformation are discussed.

Preservation of resuspended red cell concentrates. Rate of vesiculation and of spontaneous hemolysis.

In resuspended red cell concentrates addition of sucrose, mannitol and sorbitol (30 mM final concentration each) to the SAG medium (150 mM NaCl, 50 mM glucose, 1.25 mM adenine) results in a significant reduction of the spontaneous hemolysis of the cells to about 25% after 3 weeks and to about 40% after 6 weeks preservation. Furthermore, in comparison to the SAG medium the vesiculation rate is reduced to about 40% after 3 weeks preservation. Clear cut differences in the effects between the three additives could not be found. The addition of guanosine (1.25 mM final concentration) to the SAG-sucrose or SAG-sorbitol medium has no significant effects on hemolysis and vesiculation.

Membrane skeleton alteration--a factor promoting IgG receptor expression in the erythrocyte membrane.

IgG receptor expression after selective cross-linking of spectrin by means of diamide was investigated. A diamide concentration dependent IgG loading of erythrocytes was observed. Furthermore, diamide causes disturbance of lipid asymmetry, decrease of the anisotropy after topooptical staining of the glycocalyx, increase of the phagocytosis index and aggregation of the IMP's. Our findings support the hypothesis that the arrangement of the membrane skeleton at the inner aspect of the membrane is decisive not only for the lipid asymmetry but also for the spatial structure of the glycocalyx at the outer aspect of the membrane and thus for the degree of exposure and arrangement of IgG-receptors, which are thought to be localized at an extracellular portion of band 3 protein.

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran.

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.

Red blood cell aging--membrane skeleton alteration and IgG receptor expression.

Investigations were performed on aging of erythrocytes. It has been assumed that structural changes of the membrane result after exposer of the cells to certain environmental influences in vivo or in vitro. Cell aging can be connected with varying combinations of membrane structure disturbances. It is postulated that the messenger which signals membrane structure lesion is involved in a mechanism given by the expression of immunoglobulin G (IgG) receptor sites which bind autologous IgG1 and IgG3. This antibodies are cytophilic for macrophages. The performed studies demonstrated that an intact molecular arrangement of the membrane skeleton is not only a supposition for stabilization of the membrane asymmetry but also for IgG receptor masking to prevent an early elimination of the red blood cells from the organism.

The rate of lateral diffusion of phospholipids in erythrocyte microvesicles.

31P-NMR spectra of phospholipids in membranes of erythrocyte microvesicles isolated from outdated blood units were recorded in the temperature range 5 to 55 degrees C. Within that range the lineshape is strongly influenced by an increasing rate of lateral diffusion of phospholipids. At 36 degrees C a diffusion constant, D, of (2 +/- 1) X 10(-12) m2/s was obtained. The diffusion rate is by a factor of 3 to 10 greater than in erythrocyte membranes measured by the photobleaching technique and is comparable with values obtained for several lipid model membranes. The differences in lateral diffusion rates are probably connected with the depletion of microvesicle membranes in membrane proteins.

Topo-optical investigations of the conformational change of the erythrocyte glycocalyx in dependence on extracellular pH and presence of dextran.

The erythrocyte (RBC) glycocalyx reacts very sensitively to environmental influences. But few is known about the effects of certain factors, which can change the membrane structure. Therefore we have tried to investigate in model experiments by means of the topo-optical toluidine blue reaction the conformational changes of the RBC glycocalyx in dependence on pH (range from 7.5 to 4.5) and presence of dextran. The findings, which are presented here, demonstrate a high sensitivity of the glycocalyx to pH influences. A distinct drop of the membrane anisotropy can be observed in the pH range from 6.5 to 4.5. Also dextran molecules evidently cause structural changes of the RBC glycocalyx via interaction with H-bonds. On the basis of a hypothetical structural model of the glycophorins outer segment the conformational effects are discussed.

Membrane skeleton and diminution of transmembrane proteins and calmodulin in erythrocytic vesicles.

During preservation of erythrocytes vesicles are formed, the membranes of which do not contain membrane skeleton components (spectrin, actin). In comparison to the normal erythrocyte membrane (100%) the vesicle membrane exhibits significantly lowered amounts in band 3 protein (14-17%), sialic acid (30-40%), (Ca2+ + Mg2+)ATPase (6%) and (Na+ + K+)ATPase (28%). The losses in band 3 protein, glycophorin A (calculated from the decrease of sialic acid) and in the number of the intramembranous particles correspond to one another. The calmodulin concentration of the vesicles was estimated to be 35% that of the erythrocytes in maximum. The findings indicate a binding of these components to the membrane skeleton. The nature of the binding of the transmembrane proteins as well as the consequences of these results regarding the composition of the intramembranous particles are discussed.

[Structure of the erythrocyte membrane]. / Die Struktur der Erythrozytenmembran.

A short survey is given about the components and the structure of the human erythrocyte membrane. The dominating transmembrane proteins, the anion exchange protein as a main constituent of the intramembranous particles and as an anchoring site of the membrane skeleton, and the glycophorin A as the main component of the glycocalyx are discussed in particular. The structure of the membrane skeleton, its binding sites at the inner membrane aspect as well as the nature of the junctions of that network and the functions of the membrane skeleton are spoken about.

Activation of acetylcholine esterase (ACHE) as a sign for erythrocyte membrane alteration.

In vitro activity determination of membrane bound acetylcholine esterase (ACHE) of intact erythrocytes and differently altered RBC forms by means of the ELLMAN method yielded increase in activity in erythrocyte/vesicle mixtures and ghosts. Alteration of the cells by treatment with periodate, neuraminidase and trypsin leads to no, weak or clear decrease in the activity of this enzyme. Old cells also show a clear decrease in activity. When cells were treated with diamide ACHE is at first functionally inhibited, finally, however, it reaches supernormal values of activity. Possible causes for the different behaviour of ACHE in dependence on the degree of alteration of the erythrocytes are discussed. The results of the measurements were compared with the ultrahistochemical ACHE reactions according to KARNOVSKY (thiocholine--copper ferricyanide method) and IWAYAMA (lead thiocholine method). The influence of several components of the KARNOVSKY'S histochemical reaction mixture on ACHE activity was tested. The investigations suggest that disintegration of the erythrocyte membrane is accompanied with activation of the ACHE molecules which are nearly inactive in the intact membrane. This activation contributes to the positive histochemical ACHE test, which is an indicator reaction of membrane disintegration. Nearly intact membranes show a negative reaction. On the basis of these results we could show that physiological enucleation of normoblasts is connected with the formation of disintegrated membrane areas and their discharge from the future reticulocyte membrane.

Topo-optical investigations of the human erythrocyte glycocalyx-age related changes.

The conformational state of the glycocalyx of the intact and altered erythrocyte membrane was studied by means of the topo-optical toluidine blue reaction, i.e. induced membrane birefringence. High membrane anisotropy represents the normal glycocalyx structure and its decline represents their perturbation. The results show that the glycocalyx structure is changed during ageing of the erythrocytes in vivo as well as in vitro. During fluid preservation, in vitro ageing and vesiculation of cells in vitro, a subpopulation of cells showed a decline of membrane anisotropy, but other cells demonstrated abnormally high values. In the latter cases, there is usually a correlation to spherocytes. From this point of view, it is to be assumed that spherogenesis during cell ageing is induced by cell vesiculation. This leads to a remodelling of an intact plasmalemma. In contrast, the cell fractions which are probably non-vesiculating seem to be more or less damaged by membrane and/or plasmic hydrolases. This can be mimicked by neuraminidase and protease treatment of erythrocytes in vitro. Membrane lesions caused by freeze preservation of red blood cells are rare. The topo-optical results are interpreted according to the assumptions of the theory of membrane anisotropy, i.e. the formation of dye-stuff micelles at distinct, clustered, sialylated carbohydrate chains of the glycophorin A.

The histochemical detection of acetylcholine esterase--an indicator of the disintegration of membranes.

The presence of ACHE in erythrocytes membrane in vitro but not detectable ultrahistochemically has been explored as a sign of membranes disintegration. The final results of the investigations have shown, that the increase in ACHE activity of the erythrocyte membrane is a qualitative sign and a semiquantitative measure of serious disturbances in the membrane structure.