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Membrane nanotubes (NTs) are dynamic communication channels connecting spatially separated cells even over long distances and promoting the transport of different cellular cargos. NTs are also involved in the intercellular spread of different pathogens and the deterioration of some neurological disorders. Transport processes via NTs may be controlled by cytoskeletal elements. NTs are frequently observed membrane projections in numerous mammalian cell lines, including various immune cells, but their functional significance in the 'antibody factory' B cells is poorly elucidated. Here, we report that as active channels, NTs of B-lymphoma cells can mediate bidirectional mitochondrial transport, promoted by the cooperation of two different cytoskeletal motor proteins, kinesin along microtubules and myosin VI along actin, and bidirectional transport processes are also supported by the heterogeneous arrangement of the main cytoskeletal filament systems of the NTs. We revealed that despite NTs and axons being different cell extensions, the mitochondrial transport they mediate may exhibit significant similarities. Furthermore, we found that microtubules may improve the stability and lifespan of B-lymphoma-cell NTs, while F-actin strengthens NTs by providing a structural framework for them. Our results may contribute to a better understanding of the regulation of the major cells of humoral immune response to infections.

Membrane nanotubes are cell protrusions that grow to tens of micrometres and functionally connect cells. Actin filaments are semi-flexible polymers, and their polymerisation provides force for the formation and growth of membrane nanotubes. The molecular bases for the provision of appropriate force through such long distances are not yet clear. Actin filament bundles are likely involved in these processes; however, even actin bundles weaken when growing over long distances, and there must be a mechanism for their regeneration along the nanotubes. We investigated the possibility of the formation of periodic molecular relay stations along membrane nanotubes by describing the interactions of actin with full-length IRSp53 protein and its N-terminal I-BAR domain. We concluded that I-BAR is involved in the early phase of the formation of cell projections, while IRSp53 is also important for the elongation of protrusions. Considering that IRSp53 binds to the membrane along the nanotubes and nucleates actin polymerisation, we propose that, in membrane nanotubes, IRSp53 establishes molecular relay stations for actin polymerisation and, as a result, supports the generation of force required for the growth of nanotubes.

Introduction: Galectins are master regulators of maternal immune responses and placentation in pregnancy. Galectin-13 (gal-13) and galectin-14 (gal-14) are expressed solely by the placenta and contribute to maternal-fetal immune tolerance by inducing the apoptosis of activated T lymphocytes and the polarization of neutrophils toward an immune-regulatory phenotype.Furthermore, their decreased placental expression is associated with pregnancy complications, such as preeclampsia and miscarriage. Yet, our knowledge of the immunoregulatory role of placental galectins is incomplete., Methods: This study aimed to investigate the effects of recombinant gal-13 and gal-14 on cell viability, apoptosis, and cytokine production of peripheral blood mononuclear cells (PBMCs) and the signaling pathways involved., Results: Herein, we show that gal-13 and gal-14 bind to the surface of non-activated PBMCs (monocytes, natural killer cells, B cells, and T cells) and increase their viability while decreasing the rate of their apoptosis without promoting cell proliferation. We also demonstrate that gal-13 and gal-14 induce the production of interleukin (IL)-8, IL-10, and interferon-gamma cytokines in a concentration-dependent manner in PBMCs. The parallel activation of Erk1/2, p38, and NF-ĸB signaling evidenced by kinase phosphorylation in PBMCs suggests the involvement of these pathways in the regulation of the galectin-affected immune cell functions., Discussion: These findings provide further evidence on how placenta-specific galectins assist in the establishment and maintenance of a proper immune environment during a healthy pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oravecz, Romero, Tóth, Kapitány, Posta, Gallo, Rossi, Tarca, Erez, Papp, Matkó, Than and Balogh.)

Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.

The positive coreceptor function of complement receptor type 2 [CR2 (CD21)] on B cells is generally accepted, although its role in the enhancement of antibody production had only been proven in mice. The importance of this phenomenon prompted reinvestigation of the functional consequences of coclustering CD21 and the B cell receptor (BCR) on primary human cells. We found that, at non-stimulatory concentrations of anti-IgG/A/M, coclustering the BCR and CR2 enhanced the Ca 2+ response, while activation marker expression, cytokine production, proliferation, and antibody production were all inhibited upon the coengagement of CR2 and BCR on human B cells. Thus, the "textbook dogma" claiming that C3d acts as an adjuvant to enhance humoral immunity is relevant only to mice and not to humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kovács, Mácsik-Valent, Matkó, Bajtay and Erdei.)

Nanotubular connections between mammalian cell types came into the focus only two decades ago, when "live cell super-resolution imaging" was introduced. Observations of these long-time overlooked structures led to understanding mechanisms of their growth/withdrawal and exploring some key genetic and signaling factors behind their formation. Unbelievable level of multiple supportive collaboration between tumor cells undergoing cytotoxic chemotherapy, cross-feeding" between independent bacterial strains or "cross-dressing" collaboration of immune cells promoting cellular immune response, all via nanotubes, have been explored recently. Key factors and "calling signals" determining the spatial directionality of their growth and their overall in vivo significance, however, still remained debated. Interestingly, prokaryotes, including even ancient archaebacteria, also seem to use such NT connections for intercellular communication. Herein, we will give a brief overview of current knowledge of membrane nanotubes and depict a simple model about their possible "historical role"., (© 2021. The Author(s).)

IL-10 is a suppressive cytokine that has been implicated in the pathophysiology of autoimmune disorders and can be produced by different cell types such as regulatory B-cells. Our previous work showed that under inflammatory condition MZ B-cells differentiated into IL-10 producing cells and contributed to the downregulation of collagen-induced arthritis, while follicular B-cells failed to do so. Based on these observations, we aimed to investigate how inflammatory signals mediated through the BCR, TLR9 and IFN-γ receptors trigger IL-10 production in MZ B-cells but leave FO B-cells unresponsive. We particularly focused on the CREB transcription factor as it is involved in all three signalling cascades and analysed its contribution to IL-10 production. Our results demonstrate that the IL-10 production of MZ B-cells induced by the BCR, TLR9 and IFN-γ receptors is mediated by CREB. We showed that the activation of CREB is prolonged in MZ B-cells while the transcription factor only transiently phosphorylated in FO B-cells. The sustained phosphorylation of CREB is clearly associated with its prolonged binding to molecular partner CBP, whereas inhibition of their association decreased IL-10 production. We assume that sustained activation of CREB is required for IL-10 production by B-cells under inflammatory conditions., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication. These tethers can form spontaneously between many cell types, including cells of the immune and nervous systems. Traffic of viral proteins, vesicles, calcium ions, mRNA, miRNA, mitochondria, lysosomes and membrane proteins/raft domains have all been reported so far via the open ended tunneling nanotubes (TNTs). Recently we reported on existence of plasma membrane derived GM 1 /GM 3 ganglioside enriched microvesicles and costimulatory proteins in nanotubes connecting B lymphocytes, the way they are formed and transported across TNTs, however, still remained unclear. Here, using live cell confocal and Structured Illumination (SR-SIM) superresolution imaging, we show that B cells respond to bacterial (Cholera) toxin challenge by their subsequent internalization followed by rapid formation of intracellular microvesicles (MVs). These MVs are then transported between adjacent B cells via nanotubes. Selective transport-inhibition analysis of two abundant motor proteins in these cell types demonstrated that actin-based non-muscle myosin 2A dominantly mediates intercellular MV-transport via TNTs, in contrast to the microtubule-based dynein, as shown by the unchanged transport after inhibition of the latter. As suggested by SR-SIM images of GFP-CD86 transfected macrophages, these costimulatory molecules may be transferred by unusually shaped MVs through thick TNTs connecting macrophages. In contrast, in B cell cultures the same GFP-CD86 is dominantly transported along the membrane wall of TNTs. Such intercellular molecule-exchange can consequently improve the efficiency of antigen-dependent T cell activation, especially in macrophages with weak costimulator expression and T cell activation capacity. Such improved T cell activating potential of these two cell types may result in a more efficient cellular immune response and formation of immunological memory. The results also highlight the power of superresolution microscopy to uncover so far hidden structural details of biological processes, such as microvesicle formation and transport.

C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system., (Copyright © 2017. Published by Elsevier B.V.)

Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

Background: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored., Methods: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied., Results: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle., Conclusions: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Galectins are glycan-binding proteins that regulate innate and adaptive immune responses, and some confer maternal-fetal immune tolerance in eutherian mammals. A chromosome 19 cluster of galectins has emerged in anthropoid primates, species with deep placentation and long gestation. Three of the five human cluster galectins are solely expressed in the placenta, where they may confer additional immunoregulatory functions to enable deep placentation. One of these is galectin-13, also known as Placental Protein 13 (PP13). It has a "jelly-roll" fold, carbohydrate-recognition domain and sugar-binding preference resembling other mammalian galectins. PP13 is predominantly expressed by the syncytiotrophoblast and released from the placenta into the maternal circulation. Its ability to induce apoptosis of activated T cells in vitro, and to divert and kill T cells as well as macrophages in the maternal decidua in situ, suggests important immune functions. Indeed, mutations in the promoter and an exon of LGALS13 presumably leading to altered or non-functional protein expression are associated with a higher frequency of preeclampsia and other obstetrical syndromes, which involve immune dysregulation. Moreover, decreased placental expression of PP13 and its low concentrations in first trimester maternal sera are associated with elevated risk of preeclampsia. Indeed, PP13 turned to be a good early biomarker to assess maternal risk for the subsequent development of pregnancy complications caused by impaired placentation. Due to the ischemic placental stress in preterm preeclampsia, there is increased trophoblastic shedding of PP13 immunopositive microvesicles starting in the second trimester, which leads to high maternal blood PP13 concentrations. Our meta-analysis suggests that this phenomenon may enable the potential use of PP13 in directing patient management near to or at the time of delivery. Recent findings on the beneficial effects of PP13 on decreasing blood pressure due to vasodilatation in pregnant animals suggest its therapeutic potential in preeclampsia.

Elucidation of the molecular architecture of complex, highly organized molecular macro-assemblies is an important, basic task for biology. Differential polarization (DP) measurements, such as linear (LD) and circular dichroism (CD) or the anisotropy of the fluorescence emission (r), which can be carried out in a dichrograph or spectrofluorimeter, respectively, carry unique, spatially averaged information about the molecular organization of the sample. For inhomogeneous samples-e.g. cells and tissues-measurements on macroscopic scale are not satisfactory, and in some cases not feasible, thus microscopic techniques must be applied. The microscopic DP-imaging technique, when based on confocal laser scanning microscope (LSM), allows the pixel by pixel mapping of anisotropy of a sample in 2D and 3D. The first DP-LSM configuration, which, in fluorescence mode, allowed confocal imaging of different DP quantities in real-time, without interfering with the 'conventional' imaging, was built on a Zeiss LSM410. It was demonstrated to be capable of determining non-confocally the linear birefringence (LB) or LD of a sample and, confocally, its FDLD (fluorescence detected LD), the degree of polarization (P) and the anisotropy of the fluorescence emission (r), following polarized and non-polarized excitation, respectively (Steinbach et al 2009 Acta Histochem.111 316-25). This DP-LSM configuration, however, cannot simply be adopted to new generation microscopes with considerably more compact structures. As shown here, for an Olympus FV500, we designed an easy-to-install DP attachment to determine LB, LD, FDLD and r, in new-generation confocal microscopes, which, in principle, can be complemented with a P-imaging unit, but specifically to the brand and type of LSM.

B cells acquire membrane-bound cognate antigens from the surface of the APCs by forming an IS, similar to that seen in T cells. Recognition of membrane-bound antigens on the APCs initiates adhesion of B lymphocytes to the antigen-tethered surface, which is followed by the formation of radial lamellipodia-like structures, a process known as B cell spreading. The spreading response requires the rearrangement of the submembrane actin cytoskeleton and is regulated mainly via signals transmitted by the BCR. Here, we show that cytoplasmic calcium is a regulator of actin cytoskeleton dynamics in B lymphocytes. We find that BCR-induced calcium mobilization is indispensible for adhesion and spreading of B cells and that PLCγ and CRAC-mediated calcium mobilization are critical regulators of these processes. Measuring calcium and actin dynamics in live cells, we found that a generation of actin-based membrane protrusion is strongly linked to the dynamics of a cytoplasmic-free calcium level. Finally, we demonstrate that PLCγ and CRAC channels regulate the activity of actin-severing protein cofilin, linking BCR-induced calcium signaling to the actin dynamics.

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15-40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4(+) T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors' (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4(+) cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

Objective: To investigate placental protein 13 (PP13) localization in relation to cytoskeleton and lipid rafts in preeclampsia and HELLP syndrome., Study Design: Placental cryosections from patients with preeclampsia and HELLP, and controls were stained for PP13, actin, PLAP (lipid raft marker), and CD71 (nonraft marker). BeWo cells exposed to stress conditions were stained for PP13 and actin. Protein localizations were investigated by confocal microscopy, PP13 concentrations by ELISA., Results: PP13-actin colocalization was increased in syncytiotrophoblast juxtamembrane regions in term/preterm preeclampsia and HELLP. PP13-CD71 colocalization was decreased and PP13-PLAP proximity was increased in preterm but not term preeclampsia and HELLP. PP13-release from BeWo cells was inhibited by cytoskeleton disruption, and augmented by Ca2+-influx and ischemic stress., Conclusion: The actin cytoskeleton, probably in connection with lipid rafts, controls trophoblastic "nonclassical" PP13 export. PP13 is released from the syncytiotrophoblast in preterm preeclampsia and HELLP, mimicked in BeWo cells by ischemic stress, suggesting PP13 is a placental alarmin., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Endothelial cells play a critical role in inflammation by responding to several endogenous and exogenous proinflammatory stimuli. The three most studied factors that provide inflammatory signals to endothelial cells are lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β; however, their effects on endothelial cells were thoroughly compared at the level of gene expression only. Therefore, our aim was to assess the differences in the signaling pathways, adhesion molecules, and cytokines induced by proinflammatory factors in human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that signaling of LPS was less effective than that of IL-1β, and was significantly slower than that ofTNF-α and IL-1β, which can be partially explained by the special localization of Toll-like receptor 4 (TLR4). We showed that TLR4 is mainly localized in Golgi apparatus in HUVEC. The proinflammatory capacity of TNF-α was similar to that of IL-1β in inducing NF-κB nuclear translocation, while IL-1β was the strongest activator of MAPK pathways. Moreover, expression of E-selectin, IL-6, and IL-8 was induced most efficiently by IL-1β, while LPS and TNF-α had less effect, whereas we did not find such a difference in ICAM-1 and MCP-1 expression. Due to the higher induction of E-selectin and IL-8, IL-1β might have more important role in neutrophil recruitment than LPS and TNF-α. By above-mentioned parameters we identified a signaling and expression pattern for the three proinflammatory molecules. This pattern illustrates how complex a proinflammatory process can be, and may enable us to predict and compare the pathomechanism of various inflammatory diseases., (Copyright © 2010 International Society for Advancement of Cytometry.)

Estrogen plays a critical regulatory role in the development and maintenance of immunity. Its role in the regulation of antibody synthesis in vivo is still not completely clear. Here, we have compared the effect of estrogen on T cell-dependent (TD) and T cell-independent type 2 (TI-2) antibody responses. The results provide the first evidence that estrogen enhances the TD but not the TI-2 response. Ovariectomy significantly decreased, while estrogen re-administration increased the number of hapten-specific IgM- and IgG-producing cells in response to TD antigen. In vitro experiments also show that estrogen may have a direct impact on B and T cells by inducing rapid signaling events, such as Erk and AKT phosphorylation, cell-specific Ca(2+) signal, and NFkappaB activation. These non-transcriptional effects are mediated by classical estrogen receptors and partly by an as yet unidentified plasma membrane estrogen receptor. Such receptor- mediated rapid signals may modulate the in vivo T cell-dependent immune response.

The evolutionarily developed microdomain structure of biological membranes has gained more and more attention in the past decade. The caveolin-free "membrane rafts," the caveolin-expressing rafts (caveolae), as well as other membrane microdomains seem to play an essential role in controlling and coordinating cell-surface molecular recognition, internalization/endocytosis of the bound molecules or pathogenic organisms and in regulation of transmembrane signal transduction processes. Therefore, in many research fields (e.g. neurobiology and immunology), there is an ongoing need to understand the nature of these microdomains and to quantitatively characterize their lipid and protein composition under various physiological and pathological conditions. Flow and image cytometry offer many sophisticated and routine tools to study these questions. In this review, we give an overview of the past efforts to detect and characterize these membrane microdomains by the use of classical cytometric technologies, and finally we will discuss the results and perspectives of a new line of raft cytometry, the "high throughput screening assays of membrane microdomains," based on "lipidomic" and "proteomic" approaches., ((c) 2008 International Society for Advancement of Cytometry)

Lipid rafts are cholesterol- and glycosphingolipid-rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy-phosphatidylcholine was imaged with differential polarization CLSM (DP-CLSM). Mobility of the diI probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI-linked protein markers of rafts and a new anti-cholesterol antibody (AC8) in various immune cells. On the same cells, albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP-CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property--in accordance with diI mobility assessed by FCS--was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes--G(M1) and a new anti-cholesterol antibody--equivocally label the membrane rafts on a variety of cell types, while some raft-associated proteins (MHC-II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP-CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts., (Copyright 2007 International Society for Analytical Cytology.)

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Ceramide is a widely accepted mediator of T cell apoptosis and is released upon receiving various death or stress signals. Recently we have shown that the fate of T cells, life or death, depends strictly on the strength and duration of the ceramide-generating stimulus. Subapoptotic ceramide signals were shown to negatively regulate the antigen-specific activation signaling in T cells. Here we show that these subapoptotic ceramide signals also inhibit the antigen-triggered Ca2+ signals in B lymphocytes or the FcepsilonRI-mediated response of mast cells to antigen, but in a differential manner. Burkitt B lymphoma cells, frequently used models of mature B cells, and marginal zone B cells were largely resistant to the inhibitory action of ceramide. The response to cell death-inducing (strong/long duration) ceramide stimuli, resulting in massive apoptosis in T cells, was also differential among the various immunocytes in terms of both the death mechanism and the sensitivity. Our data suggest that ceramide's effects on life and death signaling in immunocytes are cell type-/stage-specific.

The mechanism of apoptosis induced by human galectin-1, a mammalian beta-galactoside-binding protein with a remarkable cytotoxic effect on activated peripheral T cells and tumor T cell lines has been extensively investigated in this study. Here we first show that galectin-1 initiate the acid sphingomyelinase mediated release of ceramide and this event is critical in the further steps. Elevation of ceramide level coincides with exposure of phosphatidylserine on the outer cell membrane. The downstream events, decrease of Bcl-2 protein amount, depolarization of the mitochondria and activation of the caspase 9 and caspase 3 depend on production of ceramide. All downstream steps, including production of ceramide, require the generation of membrane rafts and the presence of two tyrosine kinases, p56(lck) and ZAP70. Based on our findings we suggest a model of the mechanism of galectin-1 triggered cell death.

The possible regulatory mechanisms by which glycosphingolipid- and cholesterol-rich membrane microdomains, caveolar and non-caveolar lipid rafts, control the immune response are continuously expanding. In the present overview we will focus on how these membrane-organizing lipids are involved, in collaboration with tetraspanin proteins, in the formation of distinct MHC-I and MHC-II microdomains at the cell surface and will analyze the possible roles of MHC compartmentation in the processes of antigen presentation and regulation of various stages of the cellular immune response. Some basic, lipid raft- and tetraspan mediated mechanisms involved in the formation and function of immunological synapses between various APCs and T-cells will also be discussed. Finally, a new aspect of immune regulation by sphingolipids will be briefly described, namely how can the death or stress signals, leading to ceramide accumulation, result in raft-associated regulatory platforms controlling cell death or antigen-induced, TCRmediated signaling of T-lymphocytes. The influence of these signals and their cross-talk on the fate (death or survival) of T-cells and the outcome of T-cell response will also be discussed.

Sphingomyelinase (SMase)-mediated release of ceramide in the plasma membrane of T-lymphocytes induced by different stimuli such as ligation of Fas/CD95, irradiation, stress, inflammation or anticancer drugs primarily involves mitochondrial apoptosis signaling, but under specific conditions non-apoptotic Fas-signaling was also reported. Here we investigated, using a quantitative simulation model with exogenous C2-ceramide (and SMase), the dependence of activation and fate of T-cells on the strength and duration of ceramide accumulation. A murine, influenza virus hemagglutinin-specific T-helper cell (IP12-7) alone or together with interacting antigen presenting B-cells (APC) was used. C2-ceramide induced apoptosis of TH cells above a 'threshold' stimulus (>25 microM in 'strength' or >30 min in duration), while below the threshold C2-ceramide was non-apoptotic, as confirmed by early and late apoptotic markers (PS-translocation, mitochondrial depolarization, caspase-3 activation, DNA-fragmentation). The modest ceramide stimuli strongly suppressed the calcium response and inhibited several downstream signal events (e.g. ERK1/2-, JNK-phosphorylation, CD69 expression or IL-2 production) in TH cells during both anti-CD3 induced and APC-triggered activation. Ceramide moderately affected the Ca2+ -release from internal stores upon antigen-specific engagement of TCR in immunological synapses, while the influx phase was remarkably reduced in both amplitude and rate, suggesting that the major target(s) of ceramide-effects are membrane-proximal. Ceramide inhibited Kv1.3 potassium channels, store operated Ca2+ -entry (SOC) and depolarized the plasma membrane to which contribution of spontaneously formed ceramide channels is possible. The impaired function of these transporters may be coupled to the quantitative, membrane raft-remodeling effect of ceramide and responsible, in a concerted action, for the suppressed activation. Our results suggest that non-apoptotic Fas stimuli, received from previously activated, FasL+ interacting lymphocytes in the lymph nodes, may negatively regulate subsequent antigen-specific T-cell activation and thus modulate the antigen-specific T-cell response.

Background: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements., Methods: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells., Results: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format., Conclusion: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .

Peptides originally derived from complement component C3a were earlier shown to inhibit the type I FcepsilonR (FcepsilonRI)-mediated degranulation of mucosal type mast cells. In the present study, we show that C3a7, a peptide with a natural sequence, and its modified derivative, C3a9, are powerful inhibitors of the above response of both serosal and mucosal type mastocytes. We demonstrate that these peptides inhibit FcepsilonRI-induced membrane proximal events, suppress phosphorylation of the FcepsilonRI beta subunit, the protein tyrosine kinase Lyn, as well as the transient rise in free cytosolic Ca2+ level. The late phase of cellular response was also inhibited, as demonstrated by the reduced TNF-alpha secretion. Experiments using two independent methods provided evidence that the interaction site of complement-derived peptides is the FcepsilonRI beta-chain. This was further supported by fluorescence confocal microscopic colocalization and resonance energy transfer measurements. Taken together, these results suggest the presence of distinct "activating" and "inhibitory" motifs in the C3a sequence. Response to both is in balance under physiologic conditions. Furthermore, present data predict that such inhibitory peptides may serve as potent agents for future therapeutic intervention.

Background: A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,, Methods: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents., Results: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping., Conclusions: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels., (Copyright 2004 Wiley-Liss, Inc.)

Background: Lipid rafts are cholesterol- and glycosphingolipid-rich microdomains in the cellular plasma membranes that play critical roles in compartmentalization (concentration, coupling, and isolation) of receptors and signal molecules. Therefore, detecting constitutive or induced raft associations of such proteins is of central interest in cell biology. This has mostly been done with time- and cell-consuming immunobiochemical techniques affected by several sources of artifacts. A flow cytometric analysis of immunocytochemical staining under differential circumstances of detergent treatment offers a new alternative to this method., Methods: Membrane microdomains are resistant to nonionic detergents due to extensive, strong interactions between their molecular constituents. We used this feature to develop a rapid flow cytometric assay of differential detergent resistance based on immunocytochemical labeling of extracellular domain epitopes in membrane proteins. Data evaluation is based on comparative detection of their detergent solubility without and with cholesterol depletion of cell membranes, resolved by moderate concentrations of nonionic detergents., Results: Nonionic detergents Triton X-100 and Nonidet-40 (0.05-0.1%) in cold or Brij-98 (0.1-0.5%) at 37 degrees C efficiently resolved detergent solubility or resistance of many lymphocyte cell surface proteins. Kinetic data revealed that a short (5-10 min) detergent treatment is sufficient for this assay. Comparison of detergent solubility in untreated and cholesterol-depleted cells differentiated membrane proteins associated with or excluded from raft microdomains, respectively. Confocal microscopy showed that this mild detergent treatment leaves the cytoskeleton of the cells intact, with a detectable expression of raft marker detergent-resistant proteins attached to it. An induced association with rafts of immunoglobulin E receptors upon antigen cross-linking was also easily detectable in rat mast cells by this approach., Conclusions: A protocol is proposed for a rapid (5-10 min) test of detergent resistance of membrane proteins in cells. The approach requires only a small amount of cells (10(4)/sample) and offers a good resolution of detergent solubility or resistance of membrane proteins, also in terms of the underlying mechanisms, with an advantage of applicability for all conventional bench-top flow cytometers., (Copyright 2004 Wiley-Liss, Inc.)

Engagement of antigen receptors on immature B cells induces apoptosis, while at the mature stage, it stimulates cell activation and proliferation. The difference in B cell receptor (BCR)-mediated signaling pathways regulating death or survival of B cells is not fully understood. We aimed to characterize the pathway leading to BCR-driven apoptosis. Transitional immature B cells were obtained from the spleen of sublethally irradiated and auto-reconstituted mice. We have detected a short-lived BCR-driven activation of mitogen-activated protein kinases (ERK1/2 and p38 MAPK) and Akt/PKB in transitional immature B cells that correlated with the lack of c-Fos expression, reduced phosphorylation of Akt substrates and a susceptibility for apoptosis. Simultaneous signaling through BCR and CD40 protected immature B cells from apoptosis, however, without inducing Bcl-2 expression. The BCR-induced apoptosis of immature B cells is a result of the collapse of mitochondrial membrane potential and the subsequent activation of caspase-3.

Glycosphingolipid- and cholesterol-rich membrane microdomains (rafts) in T-cells are important in triggering and regulation of T(H)-cell activation in immunological synapses (IS), which in turn may control the T-cell repertoire in lymph nodes and at the periphery. It is less known, however, how the "presynaptic side" controls formation and function of IS. We investigated here activation signals and synapse formation frequency of murine IP12-7 T(H) hybridoma cell specific to influenza virus HA-peptide upon stimulation with two B-lymphoma cells, A20 and 2PK3, pulsed with peptide antigen. Confocal microscopic colocalization and FRET data consonantly revealed clustered distribution and constitutive raft-association of a major fraction of MHC-II molecules in both APCs. Costimulatory molecules (CD80 and CD86), not associated constitutively with rafts, were expressed at much lower level in A20 cells. T-cells responded to 2PK3 APC with much higher signal strength than to A20 cells, in good correlation with the frequency of IS formation, as assessed by microscopic conjugation assay. Disruption of rafts by cholesterol depletion in 2PK3 cells largely decreased the magnitude of T(H) cell activation signals, especially at low peptide antigen doses, similarly to masking CD4 with mAb on T-cells. The frequency of IS formation was reduced by blocking LFA-1 on T-cells and CD80 on APCs, by lowering the temperature below the phase transition of the membrane or by disrupting actin cytoskeleton. These data together suggest that the surface density and affinity/stability of peptide-MHC-II complexes and the costimulatory level are primary determinants for an efficient TCR recognition and the strength of the subsequent T-cell signals, as well as of the IS formation, which additionally requires a cytoskeleton-dependent remodeling of APC surface after the initial TCR signal. The threshold of T-cell activation can be further set by rafting MHC-II domains via concentrating high affinity ligands and promoting thereby T-cells for sensing low density antigen. Our data also demonstrate that B-cells, similarly to dendritic cells, could also provide T-cells with antigen-independent weak survival signals, likely associated with integrin engagement.

Diverse K(+) channels exhibit differential expression and function in human lymphocytes. Voltage-gated and Ca(2+)-activated K(+) channels have a significant impact on both T-cell activation and the overall immune response. A recent study on the plasma membrane organization of Kv1.3 K(+) channels in human T cells provides new insights into the immunoregulatory functions of K(+) channels, which might result in new and improved immune suppression protocols targeted to ion channels and/or T-cell signaling and accessory molecules.

Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous beta(2)-microglobulin (beta(2)m) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess beta(2)m resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8(+) cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of beta(2)m available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum beta(2)m levels are reported.

A theoretical analysis of experimental data is presented in this mini-review on non-random homo- and hetero-associations of cell surface receptors, which can be recruited in the plasma membrane or at the surface of the rough endoplasmic reticulum during the protein synthesis. In the latter case, the likely genetic origin of these supramolecular formations is analyzed, contrasting this concept to the mobility of the cell surface proteins. A model is offered which, on the one hand, allows the mobility in a restricted way even among microdomain-confined receptor proteins through 'swapping partners'. On the other hand, the lack of mixing molecular components of protein clusters will be analyzed, when homo-and hetero-associations are studied through cell fusion experiments. The most frequently studied cell surface patterns have included lipid raft organized HLA class I and II, ICAM-1, tetraspan molecules, IL2 and IL15 and other receptors, as well. On the contrary coated pit-associated transferrin receptors would not mix with the above lipid raft associated receptor patterns, although transferrin receptor would readily oligomerize into homo-associates. The functional consequences of these superstructures are also analyzed. On the 30th anniversary of the Singer-Nicolson fluid mosaic membrane model one has to pay tribute to the authors, because of their deep insight emphasizing also the mosaicism of the membranes in general and that of the plasma membrane, in particular.