Developmental modulation of neuronal cell surface determinants

Proceeding from the hypothesis that cellular differentiation processes are correlated with structural changes of the cell membrane, the expression of antigen and lectin receptors, as well as lectin-like molecules during migration and differentiation of pre- and perinatal neurons in the cerebral cortex, was analysed. It could be shown that a number of cell surface structures exist throughout the whole pre- and perinatal period, e.g. receptors for Robinia pseudoacacia lectin (RPL), pokeweed lectin (PWL) and concanavalin A (ConA) and the Ia and H-2-D/K antigens of the major histocompatibility complex (MHC). The expression of other cell surface structures, for example receptors for peanut and Limulus polyphemus lectin (LPL) and of Thy-1, is determined by the developmental stage; i.e. in the perinatal period higher amounts are found than in early prenatal stages. Binding sites for Phaseolus vulgaris lectin, PWL and anti-Thy-1.2 are not only demonstrated on perikaryal membranes, but additionally on diverse tangential or radial fibre structures. While on cells of the ventricular layer - the proliferating cell compartment - peanut lectin (PNL) receptors are observed in low density, LPL receptors in high density, in the migration zone, i.e. the intermediate layer, receptors are found predominantly for PNL and only few cells carry a significant number of LPL-binding sites. After the preneurons have migrated through the intermediate layer and the neighbouring cortical plate, remaining at the pia-near border of the latter, LPL receptors are again expressed on the cell surface of the now bipolar preneurons, while PNL receptors cannot be demonstrated any more. Experimental evidence is put forward indicating the possibility that this modulation of exposed carbohydrate residues on the cell surface might be mediated by a membrane-associated enzyme system on the same single molecule. For the investigation of neuronal cell interaction the ability of disintegrated suspended preneurons was used to reaggregate spontaneously in vitro and to build histiotypic cell formations within these reaggregates. It was found that this reaggregation of suspended neuronal single cells is dependent on the presence of ionized calcium, on the temperature, and on the conditions that influence the frequency of cell contacts. Furthermore, the structures expressed on the cell surface of preneurons during the pre- and perinatal period were investigated in regard to their influence on the reaggregation of these cells by means of a blockade by monoclonal antibodies or saccharides, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of cell surface lectins on activated human lymphoid cells

A cell surface lectin found on activated human lymphoid cells has been identified and characterized using membrane glycoprotein micelles as probes. These micelles, which are large, water-soluble aggregates, are composed of glycoproteins isolated from detergent-solubilized membranes of human B lymphoblastoid cell lines by Lens culinaris hemagglutinin affinity chromatography. The micelles have an average apparent molecular weight of 4 x 10(6) estimated by gel filtration and range in diameter from 25-100 nm. Micelles bind to B and T lymphoblastoid cell line cells and peripheral blood lymphocytes activated with concanavalin A or in a mixed lymphocyte response. Unactivated peripheral blood lymphocytes and red blood cells bind very low levels of the micelles. The binding is saturable, reversible, and temperature-dependent, with poor binding below 15 degrees C. Glycoproteins such as fetuin and porcine thyroglobulin, which contain complex oligosaccharide side chains, inhibit the binding, whereas glycoproteins containing only high mannose or simple serine-linked carbohydrate side chains do not. In addition, binding can be inhibited by complex asparagine-linked glycopeptides purified from pronase-digested fetuin, but not by the simple serine-linked glycopeptides. Membrane glycoprotein micelles are bound to the surface of the cells but are not internalized or degraded. The potential role of this cell surface lectin in lymphocyte function is discussed.     

Ultrastructural characterization of effector-target interactions for human neonatal and adult NK cells reveals reduced intercellular surface contacts of neonatal cells

Limitations in neonatal natural killer (NK) cell responses may be associated with the less efficient newborn capacity to solve viral infections. Although these limitations have been extensively reported they are poorly characterized. Making use of the major histocompatibility complex (MHC) class I negative cell line K562, the parameters required for the initial events involved in neonatal NK/target cell interactions were determined and compared with adult blood NK cell/target cell interactions. Ultrastructural characterization of effector-target cell interactions revealed that neonatal NK cells are more strongly activated upon contact with K562 cells than adult blood NK cells. Furthermore, the neonatal capacity to establish contacts, in particular extensive contacts, is significantly reduced when compared with adult blood NK cells. However, no significant differences were found either in the cell surface expression levels or activation state of LFA-1, which could account for the reduced intercellular contacts. Because extensive contacts are crucial for effective immunologic synapse formation, these data suggest that a limited or nonsustained positive signaling may occur on neonatal NK cells, restricting their NK cell-mediated lysis capacity.     

Expression and regulation of interleukin-33 in human monocytes

Interleukin‐33 (IL‐33) is an IL‐1 family cytokine that has a role in regulating T helper type 2 cytokines and mast cell development. Expression of IL‐33 is also associated with chronic inflammatory conditions such as rheumatoid arthritis. However, there is little information regarding IL‐33 in myeloid cell immune responses, which are important in immunity and inflammation. We therefore investigated the expression, intracellular location and regulation of myeloid cell IL‐33 by lipopolysaccharide (LPS) from Escherichia coli and the periodontal pathogen Porphyromonas gingivalis. We detected IL‐33 messenger RNA in the human promonocytic cell line THP‐1, in monocytes derived from these cells and in primary human monocytes. However, IL‐33 was not expressed in primary monocyte‐derived dendritic cells. Stimulation of monocytes with E. coli LPS (Toll‐like receptor 4 agonist) and LPS from P. gingivalis (Toll‐like receptor 2 agonist) up‐regulated IL‐33 at both the messenger RNA and protein levels but IL‐1β and tumour necrosis factor‐α had no effect. The IL‐33 protein was mainly found in the cytoplasm of monocytes with no evidence of nuclear translocation in stimulated cells. Furthermore, no IL‐33 secretion was detected after stimulation with LPS and/or ATP. These data indicate that the function, if any, of IL‐33 in activated monocytes is primarily intracellular. Interestingly, immunofluorescence analysis indicated that IL‐33 was sequestered in the nucleus of monocytes undergoing apoptosis but released into the extracellular milieu by LPS‐stimulated cells in which necrosis had been induced by freeze–thawing. Therefore, this endorses the view that IL‐33 may function as an ‘alarmin’ and have a role in signalling cellular damage and inflammatory disease pathogenesis through release from damaged or necrotic cells.     

Expression of Fc epsilon receptors and surface and cytoplasmic IgE on human fetal and adult lymphopoietic tissue

The appearance during ontogeny of IgE-positive and Fc epsilon receptor (FcER)-bearing cells was studied. Monoclonal antibody to the constant region (Fc) of IgE (CIA-E-7.12) was used to detect cytoplasmic and surface IgE. A monoclonal antibody to the low-affinity Fc epsilon receptor (FcER-II = CD23) and immune complexes composed of human IgE and mouse monoclonal anti-human IgE Fc were used to detect FcER. Cryostat sections of human fetal tissues (liver, lung, spleen, and thymus) from 11 to 22 weeks gestation as well as adult tonsil tissues were examined for IgE, FcER, and other lymphoid markers by immunoperoxidase staining. Although both IgE- and FcER-positive cells were present in adult tissues, we found that, in contrast to an earlier report, such cells were not present in the fetal tissues examined. The in situ location of FcER on cells in human lymphoid tissues revealed that the FcER-bearing cells were localized predominantly in the germinal centers (mature B cell and macrophage areas) of the tonsil follicles with some staining in the mantle (resting and less mature B cell areas).     

Distribution and scatter of yeast cell phagocytosis by human monocytes in an improved glass surface assay

The assessment of yeast cell phagocytosis by glass-adherent monocytes was improved by ultrasonication of yeast cells prior to the experiments in order to prevent aggregation, restriction of measurements to completed engulfment regardless of the number of peptides ingested, and stopping of phagocytosis before counting by maintained cooling and the addition of EDTA. The modifications provided a reduced dispersion of individual values, increased engulfment of yeast cells, and decreased numbers of yeast cells only adherent to the monocyte membrane. The improvement made the method more convenient for the study of engulfment by monocytes.     

A surface antigen marker for human monocytes.

Antisera have been raised in rabbits aginst human peritoneal macrophages.After absorption with tonsil cells the sera reacted, by direct and indirect immunfluorescence, with phagocytic mononuclear cells from a variety of tissues and, in addition, stained a small population of nonphagocytic non-T, non-B mononuclear cells present in blood, spleen, and marrow but absent or very rare in tonsil and thymus. The antisera may define a human monocyte-macrophage cell surface differentiation antigen (HuMA) and can be used to deplete or enrich reactive cell population.     

Comparison of cytotoxic properties of neonatal and adult neutrophils and monocytes and enhancement by cytokines.

We studied cytotoxic capabilities of newborn polymorphonuclear leukocytes (PMNs) and monocytes and their enhancement by cytokines and antibodies. Umbilical cord PMNs were assessed for their ability to kill various target cells spontaneously, after activation with phorbol myristate acetate, in the presence of antiserum (antibody-dependent cellular cytotoxicity), and in the presence of dually specific antibody (heteroantibody-mediated cytotoxicity). Target cells included the K562 cell line (natural killer cell target), chicken erythrocytes (CRBCs), and herpes simplex virus-infected CEM cell lines. Newborn PMNs were equivalent to adult PMNs in their cytotoxic capacity in several cytotoxicity assays. Neither adult nor newborn PMNs lyse tumor cell targets (i.e., K562 cells) spontaneously, but both lyse K562 cells following activation with phorbol myristate acetate. Both adult and newborn PMNs lyse CRBCs and herpes simplex virus-infected CEM cells in antibody-dependent cellular cytotoxicity assays, and this lysis could be enhanced by the cytokines granulocyte-macrophage colony-stimulating factor and gamma interferon. PMN heteroantibody-mediated cytotoxicity, resulting from the use of an antibody with dual specificity to CRBCs and immunoglobulin G FcRII, was greater in newborn PMNs than in adult PMNs; however, monocyte heteroantibody-mediated cytotoxicity, resulting from the use of an antibody to CRBCs and monocyte immunoglobulin G FcRI, was lower in newborn monocytes than in adult monocytes. The percentage, but not the density, of PMNs expressing FcRII was significantly reduced in newborn PMNs compared with that in adult PMNs, while the percentages and densities of FcRI expression were equivalent in newborn and adult monocytes. We conclude that the cytotoxic capability in term newborn PMNs is equivalent to that in adult PMNs, that the activity of newborn PMNs can be enhanced by antibody and/or cytokines, and that PMNs can contribute to the newborn's ability to kill virus-infected cells.     

Spontaneous decrease of CD14 cell surface expression in human peripheral blood monocytes ex vivo

In the continuous presence of bacterial lipopolysaccharide (LPS), the membrane-bound LPS receptor, CD14, exhibits a biphasic pattern of surface expression in monocytes ex vivo; an initial increase is followed by a later decrease. In the presence of LPS, ex vivo changes in monocytic CD14 cell surface expression have been consistently interpreted as the direct result of LPS exposure. There has been little consideration for the possibility of additional cell culture effects. Here, an experiment is presented to dissect the differences between LPS effects and cell culture effects on monocytic CD14 cell surface expression ex vivo. The results show that in monocytes from diluted whole blood cultures, CD14 surface expression is induced in LPS-treated samples but decreased in untreated samples. These observations suggest that the previously observed biphasic surface expression pattern of CD14 in long-term LPS-treated monocytes may be the result of a superposition of LPS-induced expression and spontaneous disappearance of CD14 from the plasma membrane. Further, these results illustrate the importance of taking cell culture conditions into account when analyzing monocyte expression of CD14.     

Leishmania interaction with human monocytes and neutrophils: modulation of the chemotactic response

Infection of host cells with Leishmania, which are obligate parasites of mononuclear phagocytes, most probably involved chemotaxis of host cells towards the parasite. We have examined the chemotactic properties of a sonicate derived from L. mexicana amazonensis promastigotes for normal human peripheral blood monocytes and neutrophils. L. m. amazonensis sonicate exhibited chemotactic activity for monocytes and neutrophils. Treatment at 65 degrees C for 30 min, enhanced the activity for neutrophils but not for monocytes, while treatment at 100 degrees C for 60 min abolished the activity. Additional studies showed that the sonicate generated chemotactic activity in serum, presumably by activating the alternative complement pathway to produce C5a, for monocytes and neutrophils. Incubation of monocytes and neutrophils with the sonicate inhibited the chemotactic activity of these cells towards various chemoattractants. When the sonicate was heat-treated the inhibitory effect was lost, except when sonicate was used as a chemoattractant. These results indicate the presence of specific receptors for factor(s) from L. m. amazonensis promastigotes on human monocytes and neutrophils.     

Giant cell formation by peripheral human monocytes

Peripheral monocytes from patients with breast carcinoma were incubated in vitro using an under agarose technique. The monocytes were incubated at 37 degrees C in a 5% CO2 atmosphere using a mixture of 0.75% agarose gel in Eagle's medium supplemented with 10% group AB pooled human serum and 50 micrograms/ml zymosan-activated serum. Significant giant cell formation occurred after 6 days incubation and was most marked after 9 days. The optimum pH for giant cell formation was between 7.0 and 7.4. The age of the subject (range 20-50 years), type of coagulant, delay in monocyte separation, storage of the monocyte preparation for up to 24 h at 4 degrees C, and freezing for up to 3 months, had no effect on giant cell formation. No giant cell formation was observed in control subjects.     

Multi‐nucleated giant cell formation from human cord blood monocytes in vitro,in comparison with adult peripheral blood monocytes

Multi‐nucleated giant cells (MGCs; Langhans‐type cell), formed from macrophage fusion, are recognized as a hallmark histological feature in chronic inflammation. However, their precise pathological role is still poorly understood, especially for microorganism pathogens in the neonatal immune system, which are capable of surviving intracellularly in phagocytes. To conduct a partial evaluation of the monocyte function of neonates, we investigated the ability of human cord blood monocytes to form MGCs in vitro by stimulating various cytokines and comparing them with adult peripheral blood monocytes. Monocytes from cord blood and adult peripheral blood were isolated and cultured for 14 days with cytokines known to induce MGC in vitro. The fusion index in experiments with a combination of interleukin (IL)‐4 and macrophage colony‐stimulating factor (M‐CSF) and a combination of IL‐4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was significantly lower in cord blood than in adult blood monocytes (P = 0·0018 and P = 0·0141, respectively). The number of nuclei per MGC was significantly lower in cord blood than in adult blood monocytes in experiments with IL‐4 alone, the combination of IL‐4 and M‐CSF, and the combination of IL‐4 and GM‐CSF (P < 0·0001). These results suggest the possibility that the susceptibility of newborns to mycobacterium infection is due partly to impaired MGC formation.     

Heterogeneity of complement receptor expression on surface immunoglobulin-bearing cells from neonatal and adult mice

The ontogeny of acquisition of complement receptors (CR) on splenic B lymphocytes from mice of varying ages was examined. Reagents used for the identification of CR included antibody- and complement-coated erythrocytes (EAC), bacteria (BAC) and bacteria treated with complement alone (BC). When whole mouse serum was used as the source of Complement both EAC and BAC failed to bind to human erythrocytes (C3b receptor-negative). showed binding to Raji and Daudi cells (C3d receptor) and formed rosettes with human neutrophils (C3bi receptor). Therefore these reagents bore C3d and C3bi, but not intact C3b. Although EAC and BAC detected nearly equal percentages of CR lymphocytes in adult mice, BAC bound to a higher percentage of neonatal lymphocytes (8-1296) than did EAC (1-395). The ability of BAC to detect EAC-negative lymphocytes among neonatal spleen lymphocytes appeared to be due to the increased sensitivity of this bacterial reagent for binding to CR. Further evidence of this enhanced sensitivity was that BAC were less inhibitable than EAC by soluble antigen-antibody-complement complexes.     

Expression of complement factor H on the cell surface of the human monocytic cell line U937

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.     

Lectin-induced modulation of snail hemocyte surface determinants: clearance of Con A-receptor complexes

The fate of surface concanavalin A (Con A)-receptor complexes on hemocytes from two stocks of the snail, Biomphalaria glabrata (PR albino and 10-R2), was studied using both direct (Con A-FITC) and indirect (biotin-Con A, avidin-FITC) fluorescent staining methods. The results of experiments in which living hemocytes were exposed first to biotin-Con A, followed by fixation and subsequent treatment with avidin-FITC to localized surface-bound lectin, demonstrate that Con A complexes are rapidly cleared from hemocyte plasma membranes. Experiments employing Con A-FITC further indicate that clearance is accomplished through an internalization process whereby Con A-complexes are endocytosed and eventually accumulate as a single intracellular aggregate of fluorescent material in the cell body of each hemocyte. In addition, a redistribution of surface complexes into patches and caps is concomitant with receptor clearance, with a maximum frequency of patching (approx. 45%) and capping (approx. 30%) occurring within 5 and 10 min., respectively, of initial Con A exposure. PR albino and 10-R2 snail hemocytes appear indistinguishable with regards to their responsiveness to Con A-binding. The close similarity in the behavior of Con A complexes or circulating hemocytes from both snail stocks further support the conclusion that Con A-reactive determinants and their associated membrane components probably represent identical molecular populations on PR albino and 10-R2 B. glabrata blood cells.     

Expression and function of NKRP1A molecule on human monocytes and dendritic cells

In this study, we analyzed the expression and function of the lymphocyte surface lectin NKRP1A on peripheral blood monocytes (Mo) or Mo and dendritic cells (DC) derived from thymic and bone marrow precursors. De novo expression of NKRP1A and CD14 molecules was detected upon culture of CD2^? CD3^? CD14^? CD16^? CD1a^? NKRP1A^? immature thymic precursors for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Under these culture conditions, by day 21, a fraction of cells had lost CD14 and acquired both CD80 (B7.1) and CD86 (B7.2) molecules. These cells displayed a DC-like morphology and were surface NKRP1A positive. CD34⁺ NKRP1A^? CD14^? precursors, isolated from bone marrow and cultured in the presence of GM-CSF, also expressed both NKRP1A and CD14: these antigens were newly expressed on about one third of cells which had lost the CD34 precursor marker. In addition, NKRP1A was constitutively present on resting CD14⁺ peripheral blood Mo. When these cells were cultured in the presence of GM-CSF, the resulting DC population retained the expression of NKRP1A and acquired CD80, while they lost the CD14 antigen. Functional analysis revealed that the engagement of NKRP1A molecule leads to a strong intracellular calcium ([Ca²⁺]_i) increase both in resting peripheral blood Mo and in vitro-derived DC. [Ca²⁺]_i increase was mainly due to extracellular calcium influx, as it was completely abrogated by the addition of EGTA. More importantly, the engagement of the NKRP1A molecule induced interleukin (IL)-1β and IL-12 production by resting Mo and DC, respectively. Altogether these data indicate that NKRP1A lectin is present at the surface of Mo and DC and may play a relevant role in the activation and function of both cell types.     

Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties

In numerous airway diseases, such as cystic fibrosis, the epithelium is severely damaged and must regenerate to restore its defense functions. Although the human airway epithelial stem cells have not been identified yet, we have suggested recently that epithelial stem/progenitor cells exist among both human fetal basal and suprabasal cell subsets in the tracheal epithelium. In this study, we analyzed the capacity of human adult basal cells isolated from human adult airway tissues to restore a well-differentiated and functional airway epithelium. To this end, we used the human-specific basal cell markers tetraspanin CD151 and tissue factor (TF) to separate positive basal cells from negative columnar cells with a FACSAria cell sorter. Sorted epithelial cells were seeded into epithelium-denuded rat tracheae that were grafted subcutaneously in nude mice and on collagen-coated porous membranes, where they were grown at the air-liquid interface. Sorted basal and columnar populations were also analyzed for their telomerase activity, a specific transit-amplifying cell marker, by the telomeric repeat amplification protocol assay. After cell sorting, the pure and viable CD151/TF-positive basal cell population proliferated on plastic and adhered on epithelium-denuded rat tracheae, as well as on collagen-coated porous membranes, where it was able to restore a fully differentiated mucociliary and functional airway epithelium, whereas viable columnar negative cells did not. Telomerase activity was detected in the CD151/TF-positive basal cell population, but not in CD151/TF-negative columnar cells. These results demonstrate that human adult basal cells are at least airway surface transit-amplifying epithelial cells.