Rapid local oscillations of the surface of the human erythrocyte

The transverse displacements of the human erythrocyte surface with amplitude 300-400 nm in the frequency range 0.2-30 Hz are recorded on the minimal area erythrocyte rim (approximately 0.5 X 0.5 microns). These local oscillations of the surface are diminished at hypoosmotic erythrocyte swelling, on addition of substances which increase the membrane rigidity (0.01% glutaraldehyde, 0.5 mM 4-hydroxymercuribenzoate, cell membrane stain--0.002% Heliogen Blue) and on discocyte--echinocyte transformation due to addition of 1-2 mM 2,4-dinitrophenol. The amplitude of transverse displacements is reduced by 1.7-2 times on erythrocytes of patients with inherent microspherocytosis. These erythrocytes have inherent defects in spectrin. It is suggested that spectrin is important for rapid local oscillations of the human erythrocyte surface.     

Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.     

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Receptor-mediated endocytosis of fibroblast β-glucuronidase by peritoneal macrophages

β-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of β-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.     

Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages.

Macrophage subpopulations in the mouse express a lectin-like receptor, sialoadhesin (originally named sheep erythrocyte receptor, SER), which selectively recognizes sialoglycoconjugates and is likely to be involved in cellular interactions of stromal macrophages in haematopoietic and lymphoid tissues. In this report we describe the purification and ligand specificity of sialoadhesin isolated from mouse spleen. Purified sialoadhesin, a glycoprotein of 185 kd apparent Mr, agglutinated sheep or human erythrocytes at nanomolar concentrations in a sialic acid-dependent manner. Low angle shadowing and electron microscopy showed that sialoadhesin consisted of a globular head region of approximately 9 nm and an extended tail of approximately 35 nm. To investigate the specificity for sialic acid, we studied the interaction of sialoadhesin with derivatized human erythrocytes, glycoproteins, and glycolipids. In conclusion, sialoadhesin specifically recognizes the oligosaccharide sequence Neu5Ac alpha 2----3Gal beta 1----3GalNAc in either sialoglycoproteins or gangliosides. These findings imply that specific sialoglycoconjugates carrying this structure may be involved in cellular interactions between stromal macrophages and subpopulations of haematopoietic cells and lymphocytes.     

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

Histidine-rich glycoprotein blocks T cell rosette formation and modulates both T cell activation and immunoregulation

Histidine-rich glycoprotein (HRGP) is a plasma and platelet protein with undefined function in vivo. It has been reported to inhibit rosette formation between murine T cells and erythrocytes. We have shown that HRGP binds specifically to human T lymphocytes but not sheep erythrocytes and have demonstrated a 56-kDa HRGP-binding protein on the T cell surface which is distinct from the CD2 sheep erythrocyte receptor. We have now investigated whether HRGP can inhibit human T cell-sheep erythrocyte rosette formation and whether HRGP can modulate T cell activation. HRGP at physiologic concentrations specifically inhibited rosette formation between human T lymphocytes and sheep erythrocytes. HRGP suppressed proliferation of antigen receptor (CD3)-triggered T cells induced by interleukin 2; this suppression was specifically reversed by prior incubation of HRGP with affinity-purified anti-HRGP IgG. Addition of HRGP 12-24 h after CD3 triggering no longer suppressed T cell proliferation, suggesting HRGP suppressed T cell division by interfering with one or more early events in the process of T cell activation. Human serum (containing 100-150 micrograms/ml HRGP) was also capable of suppressing T cell proliferation; serum which had been immunodepleted of HRGP no longer inhibited T cell proliferation. Furthermore, HRGP inhibited interleukin 2 receptor expression on activated T cells, causing decreased T cell interferon-gamma release and altered T cell-dependent inhibition of erythropoiesis. HRGP is thus capable of modulating T cell activation and T cell immunoregulation; HRGP may function as a natural suppressive regulator of human T lymphocyte activation.     

Characterization of a monoclonal antibody to guinea pig T cells that inhibits rosette formation of the cells with rabbit erythrocytes: similarity of the antigen to E-receptor on human T cells

A monoclonal antibody, GPT-1, was prepared by fusion of the splenic cells of mice immunized with guinea pig thymocytes with a mouse myeloma cell line. GPT-1 completely inhibited spontaneous rosette formation of T cells with papain-treated rabbit erythrocytes. GPT-1 reacted with 90% of thymocytes, 70% of peripheral blood lymphocytes, and 45% of splenic lymphocytes, but not with B cells. These results indicate that GPT-1 has pan-T reactivity. The antibody specifically bound to a single polypeptide chain with a molecular size of 50-65 kD. The surface density of the antigen was higher on thymocytes than on peripheral T cells, suggesting that the antigen is a certain differentiation antigen on T cells. Phytohemagglutinin-activated T cells expressed more antigen molecules than resting T cells. In addition, GPT-1 suppressed the proliferation of T cells induced by the mitogen, indicating that GPT-1 recognizes a T cell-specific surface antigen which is associated with T cell activation. Based on these results, it was concluded that GPT-1 reacts with a guinea pig T cell surface antigen which is similar to the E-receptor protein on human T cells (CD2 molecule).     

Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways

We have previously demonstrated that administration of the recently described cytokine IL-17 in rat airways in vivo recruits and activates neutrophils locally. In the current study, we examined whether endogenous IL-17 is involved in mediating neutrophil recruitment caused by endotoxin exposure in mouse airways. Our in vivo data show that local endotoxin exposure causes the release of free, soluble IL-17 protein 6 h later. Systemic pretreatment with a neutralizing anti-IL-17 Ab almost completely inhibits neutrophil recruitment 24 h, but not 6 h, after endotoxin exposure in the airways. Pretreatment with neutralizing anti-IL-6 and anti-macrophage inflammatory protein (MIP)-2 Abs inhibits neutrophil recruitment caused by local endotoxin exposure and IL-17, respectively. Our in vitro data show that endotoxin exposure stimulates the release of soluble IL-17 protein in T lymphocytes harvested from lung and spleen, respectively, and that this cytokine release requires coculture with airway macrophages. Intracellular IL-17 protein is detected in T lymphocytes from spleen but not in airway macrophages after coculture and stimulation of these two cell types. Finally, anti-IL-17 does not alter endotoxin-induced release of IL-6 and MIP-2 from T lymphocytes and airway macrophages in coculture. In conclusion, our results indicate that endotoxin exposure causes the release of IL-17 from T lymphocytes and that this cytokine release requires the presence of macrophages. Once released, endogenous IL-17 acts in part by inducing local release of neutrophil-mobilizing cytokines such as IL-6 and MIP-2, from nonlymphocyte, nonmacrophage cells, and this contributes to recruitment of neutrophils in the airways. These IL-17-related mechanisms constitute potential targets for pharmacotherapy against exaggerated neutrophil recruitment in airway disease.     

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.     

Differences in the response of several cell types to inhibition of surface receptor mobility by local concanavalina binding

Concanavalin A (conA) modulates the lateral mobility of cell surface receptors differently on different cell types. This was demonstrated by using fluorescence photobleaching recovery (FPR) to measure the inhibition of the lateral mobility of conA receptors by localized binding of conA on lymphocytes, fibroblasts, and macrophages. On mouse spleen lymphocytes, binding of conA platelets above a threshold coverage (about 12% of the upper cell-surface area) reduced the diffusion coefficient of mobile TMR-SconA-receptor complexes from 3.0×10^?10 cm²/sec to 0.6× 10^?10 cm²/sec (a 5-fold decrease), and the fraction of mobile receptors was concomitantly reduced from 0.4 to 0.11. Below the threshold occupancy, no effect on either parameter was detected. On 3T3 cells, a qualitatively similar threshold phenomenon was observed: coverage of over 9% of the upper cell surface by conA platelets induced a 3-fold reduction in the diffusion coefficient of TMR-SconA-receptor complexes from 5×10^?10 cm²/sec to 1.7× 10^?10 cm²/sec. However, no effect on the mobile fraction (about 0.4) was observed. In contrast, neither the diffusion coefficient nor the mobile fraction of TMR-SconA-receptor complexes on mouse peritoneal macrophages (both resident and thioglycolate-stimulated) or on the mouse macrophage cell line P388D₁ were affected by the binding of conA platelets in amounts covering over 50% of the upper cell surface (approx. 4.6× 10^?10 cm²/sec and 0.5 for the diffusion coefficient and mobile fraction, respectively). These differences are correlated to the different cytoskeletal functions of the various cell types studied, and are discussed regarding the mechanism of the conA-induced modulation.     

Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa

The effects of a cyclic peptide toxin, isolated from the cyanobacterium Microcystis aeruginosa, on cell morphology and ion transport in human erythrocytes, isolated rat hepatocytes and mouse fibroblasts (3T3) were studied. Neither in erythrocytes nor in fibroblasts did the toxin cause morphological alterations. In hepatocytes the toxin induced marked morphological alterations at a concentration of about 50 nM. In erythrocytes and fibroblasts no effects on ion transport were observed. In hepatocytes the toxin induced a significant increase in both phosphate and potassium efflux at concentrations far below the concentration causing morphological alterations (0.1 and 1 nM, respectively). It is suggested that the cytotoxicity of the toxin is not due to a non-specific interaction with the plasma membrane and that the effects of the toxin in hepatocytes are probably due to an interaction of the toxin with cytoskeletal elements.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Enhancement of the functional activities of human T cells after their interaction with SRBC

We have examined the functional consequences of the exposure of human lymphocytes to sheep red blood cells (SRBC). Peripheral blood lymphocytes (PBL) were incubated wih SRBC under optimal conditions for their interaction and, after lysis of the erythrocytes, the unfractionated PBL were examined for several T cell functions. Only after exposure to SRBC, but not after incubation with mouse, ox, chicken, or human erythrocytes, the unfractionated PBL showed an increased reactivity in the following functions: 1) Production of T cell growth factor, after PHA stimulation; 2) mitogenic response to suboptimal doses of PHA and Con A, and 3) response in mixed lymphocyte culture. Other functional activities, such as natural cytotoxicity (NK) and antibody-dependent cellular cytotoxicity (ADCC) were also enhanced by the interaction of PBL with SRBC, but the increases in cytotoxic activities were not consistently detected. Taken together, these results indicate that the interaction of PBL with SRBC has functional consequences in the reactivity of T cells producing an enhancement of several in vitro T cell functions.     

The xenogeneic effect. I. Antigen and mitogen-stimulated human lymphocytes produce a non-antigen-specific factor which reconstitutes the antibody response of T cell-deficient mouse spleen cells.

Modified Marbrook culture vessels with two chambers separated by a 0.2-mu porosity membrane have been utilized to show that antigen-stimulated human lymphocytes produce a soluble factor(s) which restores the ability of thymectomized, irradiated, and bone marrow-protected mice to mount a primary IgM plaque-forming cell response in vitro. In the initial experiments, the human lymphocytes plus antigen (sheep erythrocytes) were cultured in the lower chambers of the Marbrook vessels and the T cell-deficient mouse spleen cells plus sheep erythrocytes were cultured in the upper chambers. The response of the spleen cells was shown to be enhanced as a function of the number of human lymphocytes in the lower chambers. In subsequent experiments, the human lymphocytes were challenged with allogeneic lymphocytes or activated with a variety of T cell mitogens. Supernatants from these cultures, when placed in the lower chambers of the Marbrook vessels, were also capable of reconstituting the antibody-forming cell response of the mouse B cells. The results of the experiments are discussed in relation to a model of B cell induction which incorporates a non-antigen-specific "helpher" T cell.     

Effects of intravenous silica on immune and non-immune functions of the murine host.

Silica, an agent toxic for macrophages, administered i.v. to DBA/2 mice rapidly depresses the clearance of colloidal carbon by the reticuloendothelial system and reduces the in vitro phagocytic activity of peritoneal macrophages harvested 3 days after silica injection. Silica blocks the humoral immune response to sheep erythrocytes and the cell-mediated immune response to allogeneic fibroblasts when given before antigen. Silica also induces complex alterations in spleen cell responsiveness to concanavalin A involving both local and serum factors. Silica had no significant effect on the induction of interferon by statolon or Newcastle disease virus. No unequivocal evidence was obtained that silica has a direct depressive effect on cells other that macrophages, but indirect effects on lymphocytes were produced most likely by factors released from silica-lysed macrophages. Intravenous silica may prove useful for the separation of interferon induction and immune response stimulation in studies of host resistance to infection and oncogenesis. Considerable variation exists in the immunodepressive effects of different preparations of silica.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.