The characterization of phagocytotic processes using morphometry

In electromicroscopic pictures, the percentage areas of erythrophagosomes in macrophages of the spleen were determined 2 and 5 h after application of diamide and iodoacetate treated erythrocytes. The increase in erythrophagosomes is significant if compared with controls. It is mainly seen in connection with the changes of the erythrocyte membrane caused by diamide and iodoacetate which lead to an increased IgG-binding to the erythrocyte surfaces. The overcritical IgG-load acts as an phagocytosis signal for the macrophages.     

Passage time measurement of individual and blood cells through arrayed micropores on Si3N4 membrane

A new system has been developed for determining the deformability of individual red blood cells (RBCs), simulating the passage of RBCs in capillaries. The kernel of this system was the micropore array filter with an accurately defined pattern made by semiconductor microprocessing techniques. Individual microscopic RBC images were processed in parallel through a microcomputer and its interfacing circuit. An experiment with a normal RBC from a human donor demonstrated that it could pass the circular pore filter with a diameter as small as 1.0 μm at 2 cm H₂O pressure difference. Deformability of RBCs treated with diamide or acetylphenylhidralazine was also measured, showing that the system was sufficiently sensitive to detect the deformability loss due to membrane damage or to polymerization of the cytoplasma.     

In vivo recognition and clearance of red blood cells containing phosphatidylserine in their plasma membranes

We have previously investigated the interaction of macrophages with red blood cells (RBC) displaying phosphatidylserine (PS) in their surface membranes after the transfer of the fluorescent lipid analog 1-acyl-2-[(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl] phosphatidylserine to the RBC (Tanaka, Y., and Schroit, A. J. (1983) J. Biol. Chem. 258, 11335-11343). This derivative, which is rapidly transferred to the RBC at 37 degrees C, results in the efficient binding and phagocytosis of the RBC by autologous macrophages. In the present study, we show that 51Cr-labeled RBC containing [(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminododecanoyl]phosphatidylserine (NBD-PS) are rapidly cleared from the peripheral circulation of syngeneic mice and accumulate in the liver and spleen. Fluorescence microscopy of Kupffer cells and splenic macrophages isolated from the liver and spleens of these animals revealed phagocytosed fluorescent RBC, suggesting the clearance was probably due to endocytosis of the RBC. The accumulation of these RBC in the spleen was dramatic, with approximately 30% of the injected cells localizing in this organ within 60 min. In contrast, the intravenous injection of RBC containing similar amounts of NBD-phosphatidylcholine or NBD-phosphatidylglycerol did not result in clearance which differed significantly from control (untreated) RBC populations. The observed clearance of NBD-PS-containing RBC was much different than the clearance of opsonized RBC which preferentially localized in the liver. These findings show that PS in RBC can serve as a signal for triggering their in vivo recognition and concomitant elimination from the circulation and suggest that the exposure of endogenous PS in the outer leaflet of RBC which occurs in certain pathological conditions could trigger their removal from the circulation.     

Recovery from immunesuppression in mice infected with Schistosoma japonicum by treatment of praziquantel

To learn the possible alteration of immune response, hemolytic plaque-forming cells (PFC) produced in the spleen of Schistosoma japonicum infected mice treated with Praziquantel and untreated group were counted. There was no significant difference in the immunesuppression percentage between the treated and untreated groups 1 and 3 weeks after treatment. In 5 weeks after treatment, however, the immunesuppression percentage in the treated mice was markedly reduced in comparison with that of the untreated group. Recovery from immunesuppression appears to be associated with elimination or impairment of adult worms.     

Three-dimensional analysis of erythrophagosomes in rat mesenteric lymph node macrophages

Erythrocytes extravasated into the sinuses of the rat mesenteric lymph nodes as a result of short-term clamping of the portal vein were, although autologous, phagocytized markedly by the lymph node macrophages at 1 hr after reopening of the vein. The erythrophagosomes formed in the macrophages were exposed three-dimensionally by the cellular matrix maceration method and observed with a high-resolution scanning electron microscope. These results were compared to those obtained by conventional transmission electron microscopy. The process of degradation of an erythrocyte took about 6 hr. Coated pits were formed on the erythrophagosomal membrane at the early stage, and the erythrophagosomes were degraded by two different pathways: 1) the degradative pathway by invaginations of the phagosomal membrane, through which the erythrophagosome shrank and broke into secondary lysosomes, and 2) the hemolytic degradative pathway, by which it lost its content and formed a ghost.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

Immunospecific targeting of immunoliposomes, F(ab')2 and IgG to red blood cells in vivo

In this report a model to study the fate of target cells in the blood circulation after injection of appropriate immunoliposomes is discussed. The effect of intravenous administration of antimouse RBC immunoliposomes, F(ab')2 or IgG on the fate of intravenously injected 51Cr-labelled mouse RBC (Cr-mRBC) in the mouse and, particularly, in the rat was studied. The immunoliposome was of the Fab'-MPBPE-REV type (Fab'-fragments covalently linked to reverse phase evaporation vesicles by maleimido-4-(p-phenylbutyrate)phosphatidylethanolamine). In the rat model a high blood level (80%) of the injected dose of target cells, Cr-mRBC, was maintained for several hours. The elimination by Fab'-liposomes, F(ab')2 or IgG of Cr-mRBC, and subsequent uptake into liver and spleen was dose dependent. Administration of Fab'-liposomes or F(ab')2 resulted in a preferential uptake into the spleen (above a certain dose also, but much lower, uptake into the liver was observed), while after IgG administration 51Cr-label was mainly recovered in the liver. At equal protein doses (+/- 130 micrograms) Fab'-liposomes induced a faster elimination of the Cr-mRBC and a higher uptake into the spleen than F(ab')2. The potential advantage of the use of drug-loaded immunoliposomes to eliminate target cells from the blood stream and to induce a certain pharmacological effect in the target cells, in comparison with the free antibody administration of F(ab')2 or IgG is discussed.     

Effects of membrane lipids and -proteins and cytoskeletal proteins on the kinetics of cholesterol exchange between high density lipoprotein and human red blood cells, ghosts and microvesicles.

To better understand the effects of plasma membrane lipids and proteins and the cytoskeleton on the kinetics of cellular cholesterol efflux, the effects of (1), selectively depleting either sphingomyelin (SM) or phosphatidylcholine (PC); (2), cross-linking the cytoskeleton, and (3), removing certain cytoskeletal and integral membrane proteins on radiolabelled cholesterol efflux from red blood cells (RBC) have been studied. When RBC were treated with either phospholipase A2 or sphingomyelinase C to hydrolyze either 30-40% of the PC or 40-50% of the SM, respectively, the halftimes (t1/2) for cholesterol efflux to excess HDL3 were not significantly altered, with the values being 4.4 +/- 0.8 h or 3.7 +/- 0.4 h, respectively, compared to 4.6 +/- 0.6 h for control RBC. To investigate the effects of the cytoskeleton on the rate of free cholesterol (FC) desorption from the plasma membrane, the cytoskeletal proteins were cross-linked by either heat-treatment or exposure to diamide and cholesterol efflux from ghosts of these cells was measured. Cross-linking the cytoskeletal proteins by diamide treatment resulted in no significant change in t1/2 for treated (3.6 +/- 0.6 h) compared to control (4.2 +/- 0.4 h) ghosts: this suggests that the cytoskeleton does not play a large role in modulating cholesterol efflux. To investigate the effects of membrane proteins on cholesterol efflux, RBC microvesicles, containing mainly band 3 and 4 proteins and little of the cytoskeletal proteins, such as spectrin (bands 1,2) or actin (band 5), were obtained by incubation with the ionophore A23187. With excess HDL3 present, microvesicles exhibited a t1/2 of 4.2 +/- 1.9 h (compared to the t1/2 of 4.2 +/- 0.4 h for control ghosts). The results described in this paper suggest that neither changing the SM/PC ratio in the membrane nor cross-linking the cytoskeletal proteins nor removing the cytoskeleton changes the t1/2 for cholesterol efflux to excess HDL3. Presumably, the cholesterol-phospholipid interactions are insensitive to these perturbations in membrane structure.     

Interaction with homologous erythrocytes of rat T cells which act as aggressors in the mixed lymphocyte reaction.

Substantial percentages of T-enriched spleen lymphocytes or thymocytes of inbred rats were found to form rosettes with the RBC of homologous strains. When an excess of RBC was used, essentially all of the rosette-forming subpopulation of lymphocytes was removed when the rosettes were separated by centrifugation. After depletion of the lymphocytes, reactive with RBC of one strain, most of the lymphocytes reactive with RBC of other strains could be recovered in the supernatant. A very large percentage of lymphocytes of the BN strain formed rosettes when a mixture of the RBC of five other strains was tested; the percentage was, however, somewhat lower than that predicted on the basis of complete additivity. Rosettes dissociated when warmed to 37 degrees C. The lymphocytes recovered were unable to form rosettes again. In nearly all instances, the subpopulation that formed rosettes with RBC of a given strain included essentially all of the lymphocytes that acted as aggressors against peripheral leukocytes or mytomycin C-treated thymocytes of that strain; the lymphocytes in the supernatant always retained activity as aggressors against the leukocytes of one or more other strains and retained their responsiveness to PHA. Lymphocytes recovered from rosettes, by warming to 37 degrees C, were highly reactive as aggressors in the MLR against the strain providing the RBC. Varying degrees of reactivity were noted against leukocytes of other strains.     

Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein

The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Connor & Schroit (1988) Biochemistry 27, 848-851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Similarly, treatment of RBC with 5,5'-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360-12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.     

Subcellular localization of iron and heme metabolism related proteins at early stages of erythrophagocytosis

Background

Senescent red blood cells (RBC) are recognized, phagocytosed and cleared by tissue macrophages. During this erythrophagocytosis (EP), RBC are engulfed and processed in special compartments called erythrophagosomes. We previously described that following EP, heme is rapidly degraded through the catabolic activity of heme oxygenase (HO). Extracted heme iron is then either exported or stored by macrophages. However, the cellular localization of the early steps of heme processing and iron extraction during EP remains to be clearly defined.

Methodology/Principal Findings

We took advantage of our previously described cellular model of EP, using bone marrow-derived macrophages (BMDM). The subcellular localization of both inducible and constitutive isoforms of HO (HO-1 and HO-2), of the divalent metal transporters (Nramp1, Nramp2/DMT1, Fpn), and of the recently identified heme transporter HRG-1, was followed by fluorescence and electron microscopy during the earliest steps of EP. We also looked at some ER [calnexin, glucose-6-phosphatase (G6Pase) activity] and lysosomes (Lamp1) markers during EP. In both quiescent and LPS-activated BMDM, Nramp1 and Lamp1 were shown to be strong markers of the erythrophagolysosomal membrane. HRG-1 was also recruited to the erythrophagosome. Furthermore, we observed calnexin labeling and G6Pase activity at the erythrophagosomal membrane, indicating the contribution of ER in this phagocytosis model. In contrast, Nramp2/DMT1, Fpn, HO-1 and HO-2 were not detected at the membrane of erythrophagosomes.

Conclusions/Significance

Our study highlights the subcellular localization of various heme- and iron-related proteins during early steps of EP, thereby suggesting a model for heme catabolism occurring outside the phagosome, with heme likely being transported into the cytosol through HRG1. The precise function of Nramp1 at the phagosomal membrane in this model remains to be determined.     

Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

Immunocytochemical determination of membrane-bound IgG using physiologically aged and experimentally aged erythrocytes

The expression of IgG receptor sites at the band 3 protein is important for the recognition and elimination of aged and experimentally altered erythrocytes. Membrane bound IgG was detected in different erythrocyte preparations and microvesicles by means of electron microscopic procedures (protein A-gold-, protein A-gold-silver- and anti-ferritin-sandwich-technique) and light microscopic procedures (immunofluorescence). Physiologically "old", pronase and neuraminidase as well as diamide treated erythrocytes and microvesicles demonstrated significant IgG loading. An increased IgG binding of erythrocytes treated with phenylhydrazine was only evident when higher phenylhydrazine concentrations were used. Both, the alteration of the glycocalyx (conformational changes of the external segment of the glycophorins) and the alteration of the membrane skeleton lead to an unmasking of the IgG receptor site at band 3 proteins (transmembrane effect). The result is an overcritical loading of cells with IgG molecules which initiate the elimination of the erythrocytes by macrophages of the Reticulo-Histiocytic-System.     

Y Chromosome mosaicism in pouch young of the marsupial,greater glider (Marsupialia: Petauridae)

Y chromosome elimination was studied in three tissues from 13 pouch young greater gliders. There was no sex chromosome loss from the liver, spleen or bone marrow of female pouch young ranging in age from approximately 4 to 80 days. All liver cells in a 15 day old male were XY but only 75 to 80% of the cells were found to be XY in older pouch young. The Y chromosome was eliminated from a higher percentage of spleen cells, with 45 to 50% of the cells retaining the Y chromosome in 60-100 day old animals. — Y chromosome elimination in greater gliders appears to most closely resemble the X and Y chromosome elimination system of the marsupial bandicoots. There are no clear resemblances to the kinds of sex chromosome elimination which occur in eutherian mammals.     

A study on microbiology of biological film layer in rotating biological contactors

Experimental studies were carried out for upgrading the secondary treated domestic sewage effluent using the Rotating Biological Contactors (RBC) as an unit process in the premises of the existing water reclamation plant of Satellite Centre Building Complex of Indian Space Research Organisation at Bangalore. As part of these studies, a brief study was carried out on the microbiological aspects of the biological slime layer developed in the RBC. This study included observations on the development of the biological film on wetted disc surfaces of RBC, measurement of the thickness of the biological film, a discussion on significance of biofilm thickness in substrate removal, effect of wastewater characteristics on the thickness of biofilm and determination of MLSS/MLVSS ratio of biological film obtained in RBC reactor used for upgrading the secondary effluent. The results of these studies are presented in this paper. The results of identification of species of micro-organisms predominant in the biological slime layer in the RBC used for upgradation of secondary treated effluent are discussed separately in another paper.     

Detection of IgG bound at the erythrocyte membrane by means of an immunohistochemical gold-silver technique

The binding of IgG at the erythrocyte membrane during aging plays an important role in the elimination process of the cells by the reticulohistiocytic system. By means of an indirect protein A gold- and protein A gold-silver-method bound IgG was detected immunocytochemically on physiologically aged, pronase and neuraminidase treated red blood cells (RBC). At the light and electron microscopic level an increased binding of IgG at "old" as well as enzymatic treated cells in comparison to "young" and normal RBC was noted. The silver enhancement of gold particles visualized the immunostaining and permitted the semiquantitative analysis of the RBC by a scanning microdensitometer.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

How the spleen reshapes and retains young and old red blood cells: A computational investigation

The spleen, the largest secondary lymphoid organ in humans, not only fulfils a broad range of immune functions, but also plays an important role in red blood cell’s (RBC) life cycle. Although much progress has been made to elucidate the critical biological processes involved in the maturation of young RBCs (reticulocytes) as well as removal of senescent RBCs in the spleen, the underlying mechanisms driving these processes are still obscure. Herein, we perform a computational study to simulate the passage of RBCs through interendothelial slits (IES) in the spleen at different stages of their lifespan and investigate the role of the spleen in facilitating the maturation of reticulocytes and in clearing the senescent RBCs. Our simulations reveal that at the beginning of the RBC life cycle, intracellular non-deformable particles in reticulocytes can be biomechanically expelled from the cell upon passage through IES, an insightful explanation of why this peculiar “pitting” process is spleen-specific. Our results also show that immature RBCs shed surface area by releasing vesicles after crossing IES and progressively acquire the biconcave shape of mature RBCs. These findings likely explain why RBCs from splenectomized patients are significantly larger than those from nonsplenectomized subjects. Finally, we show that at the end of their life span, senescent RBCs are not only retained by IES due to reduced deformability but also become susceptible to mechanical lysis under shear stress. This finding supports the recent hypothesis that transformation into a hemolyzed ghost is a prerequisite for phagocytosis of senescent RBCs. Altogether, our computational investigation illustrates critical biological processes in the spleen that cannot be observed in vivo or in vitro and offer insights into the role of the spleen in the RBC physiology.     

Model studies of erythrophagocytosis in vitro

Macrophages of the RHS are important for the elimination of aged and altered erythrocytes. An increase in the membrane bound autologous IgG generates a recognition signal in the plasmalemma, which triggers the RBC-phagocytosis. The IgG-receptors are buried in the band 3 proteins of the membrane. Enzymatic degradations or conformational changes of the glycocalyx are accompanied by an expression of these receptors. Similar effects were obtained after alteration of the membrane skeleton (crosslinking of the spectrin). A clustering of IgG-receptors due to interaction of the denatured hemoglobin (Heinz-bodies) with the membrane seems to be unlikely. Exovesiculation represents a process of formation of an intact membrane structure. Prolonged stay of RBC in the spleen can induce membrane altering continued by increased IgG loading.     

Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight-line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol-oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol-reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2-min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S-S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley-Liss, Inc.