Antigén-bemutató sejtek és polarizált TH-limfociták kapcsolata; kostimulációs kölcsönhatások és az immunológiai szinapszis = The antigen presenting cell - T cell contact; costimulation and the immunological synapse

Öt (1 TH1, 2 TH2 és 2 TH0) hosszútávon stabilnak bizonyult egér TH hibridómát állítottunk elő és jellemeztünk részletesen. Vizsgáltuk az IL-4, GATA-3, IFNgamma és Tbet génexpressziókat, a GM-CSF, IL-2, IL-4 és IFNgamma szekréciós képességet, a TH fenotípus stabilitását, az aktiválhatóság, a homing tulajdonság és a membrán raftok szempontjából jellemző sejtfelszíni markerek megjelenését, a kalcium-válasz kiválthatóságát és karakterisztikáját, a nyugvó és aktivációt követő fehérje foszforilációs mintázatot, valamint a membrán koleszterin tartalmának és a raftok mennyiségének összefüggéseit, továbbá a TCR raft asszociáltságát. Ezek alapján jellegzetes különbségeket mutattunk ki az eltérő fenotípusú TH sejtek között. Az antigén bemutató sejtek tulajdonságainak feltérképezése kapcsán különböző B-limfocita és makrofág eredetű sejtvonalak tulajdonságait vizsgáltuk a sejttípusra jellemző illetve az antigén bemutató kapacitás szempontjából kiemelt sejtfelszíni molekulák jelenléte és a sejt funkcionális képességei szempontjából. Megállapítottuk a membrán gangliozid (GM1, GM3) (raft)-mennyiség és az antigén bemutató valamint fagocita képesség összefüggését. A membránban a raft komponens koleszterin tartalmának vizsgálatához anti-koleszterin egér monoklonális ellenanyagokat fejlesztettünk ki, melyek nemcsak a sejtmentes és sejtes közegben történő koleszterin kimutatásra bizonyultak alkalmasnak hanem segítségükkel bizonyos koleszterin-függő sejtfunkciók modulálhatók. | Five (1 TH1, 2 TH2 és 2 TH0) mouse TH hybridomas with long-term stability were established and characterized in detail. IL-4, GATA-3, IFNgamma and Tbet gene expressions, GM-CSF, IL-2, IL-4 and IFNgamma secreting capacity, phenotypical stability, expression of cell surface markers involved in activation and homing processes or markers of the membrane rafts in correlation with the membrane cholesterol content, induction and characteristics of the calcium response, the protein tyrosine phosphorylation patterns before and after activation, and TCR recruitment into membrane rafts were investigated. Characteristic differences were found in most parameters among the TH cells of different effector phenotype. APC cell lines of B-cell and monocyte/macrophage origin were also characterized for their cell surface antigen expression in correlation with their functional features. A significant correlation was found between the raft marker membrane ganglioside (GM1, GM3) expression and the antigen presenting capacity or the phagocitic activity in macrophage cells. Cholesterol specific mouse monoclonal antibodies were generated to detect raft-associated and free cholesterol in the membrane. On the basis of our preliminary results these antibodies may also act directly as modulators of certain immune functions

Az adaptív immunválasz kialakulása és szabályozása fiziológiás és kóros körülmények között: dendritikus sejtek, hízósejtek és a komplementrendszer szerepének vizsgálata = Regulation of adaptive immune responses under normal and pathological conditions; role of dendritic cells, mastocytes and the complement system

A magasabbrendű szervezetek immunhomeosztázisát a veleszületett és az adaptív immunrendszer együtt tartja fenn; egymással szorosan összefonódva, folyamatosan együttműködve. E két rendszer sejtes és molekuláris elemei - köztük a limfociták, makrofágok, dendritikus sejtek, hízósejtek, továbbá a komplementrendszer és a különböző citokinek - számos ponton hatnak egymásra úgy a fiziológiás, mint a kóros immunfolyamatok esetében. A pályázati munka során újabb kölcsönhatásokat írtunk le e két rendszer között, megismertük az egyes folyamatok molekuláris mechanizmusát, és a veleszületett immunrendszer egyes elemeink számos új funkcióját tártuk fel. Mindezzel jelentősen hozzájárultunk a veleszületett elemek adaptív választ befolyásoló ill. irányító hatásának további megismeréséhez. A pályázati munkába hét doktoranduszt vontunk be, akik közül öten már megszerezték a PhD fokozatot. Eredményeinket tizenkét, nemzetközi szaklapban megjelent publikációban közöltük; a cikkek - IF: 49.5. | The immunehomeostasis of higher vertebrates is maintained by the constant coordination and cooperation of the elements of the innate and adaptive immune system. Their cellular and molecular constituents - such as lymphocytes, macrophages, dendritic cells, mast cells, and the complement system and various cytokines - exert their effect on each other at several points under both physiological and pathological conditions. During the period of this research grant we could get further insight into the molecular mechanisms of these interactions; we learnt more about the possible ways how innate elements may influence or direct adaptive responses, moreover, we revealed some novel functions of certain elements. Seven doctoral students have been involved in this research - five of them already defended their thesis. 12 papers have been published in peer-reviewed journals; 'IF: 49.5

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures

Introduction Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. Methods CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. Results KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. Discussion Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures. Abbreviations B, bilirubin; Bsep, bile salt export pump; CYP, cytochrome P-450; GdCl3, gadolinium(III) chloride; H/KC, hepatocyte-Kupffer-cell co-culture; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography; KC, Kupffer-cell; LPS, lipopolisacharide; Mrp, multidrug resistance-associated protein; Ntcp, sodium-dependent taurocholate transporter; PB, phenobarbital; PBS, phosphate-buffered saline; PTX, pentoxyphylline; TC, taurocholat

Jelátviteli kompartmentek és proteolózis az immunválasz, valamint az immun- és idegrendszer közötti kommunikáció szabályozásában = Signaling compartments and proteolysis in regulation of immune responses and the communication between the immune- and nervous systems

Több új immunreceptor-kölcsönhatást (FcR/CR/BCR; sejthalál-R/TCR), és több szabályozó mechanizmust azonosítottunk a lipid raftok és más membrán mikro-kompartmentek, valamint limfocita adaptor fehérjék részéről, melyek fontosak a limfociták effektor funkcióiban ill. a sejthalál folyamatában. Leírtuk az utóbbi folyamatokban kritikus miozin motorfehérje izotípusok néhány molekuláris kapcsolatának szerkezeti hátterét és szabályozási lehetőségét. Kimutattuk egyes komplement (C) faktorok és receptorok sokrétű szerepét az immunválasz szabályozásában és, hogy a belőlük származtatott peptidekkel az allergiás hízósejtválasz gátolható. Leírtuk a C-rendszer szabályozó szerepét a Sclerosis Multiplex (SM) állatmodelljében is. Kimutattuk a tripszin-szerű proteáz aktivitás szabályozó szerepét a B-sejt válaszban, és valószínűsítettük szerepüket a mielin bázikus fehérje specifikus hasításában, mely fontos eleme lehet az SM kialakulásának. Eredményeink alapját képezik szelektív immunmodulánsok kifejlesztésének autoimmun ill. allergiás kórképekben. Kimutattunk egyes immunrendszeri effektor- és idegrendszeri funkciók között fellépő, a citokin hálózat ill. steroid hormonok (ösztrogének, glukortikoidok) útján megvalósuló kommunikációs útvonalakat. Új módszereket is kifejlesztettünk (pl. antigén-targetingre alkalmas egyláncú ellenanyag konstrukciók) és a projekt támogatásával beszereztünk egy konfokális mikroszkópot, melyen több új metodikát optimalizáltunk a celluláris kommunikáció vizsgálatára. | We identified several novel immunoreceptor cross-talk elements (FcR/CR/BCR; cell death-R/TCR) and regulatory mechanisms by lipid rafts, other membrane-compartments, as well as adaptor proteins, which are essential in the lymphocytes' effector functions and cell death. Structural and regulatory aspects of some important molecular interactions of myosin motor protein isotypes, essential players in the above processes, were also described. Multiple regulatory functions of the complement system in the immune response and the inhibitory potential of C3a-derived peptides in the allergic mast cell response were identified. The regulatory potential of the complement system was also shown in an animal model of Sclerosis Multiplex (SM). We have shown that trypsin-like protease activities are involved the B cell response through formation of soluble receptors and that trypsin-4, specifically cleaving MBP, may be a critical element in development of SM. Our results form a basis for development of selective immunomodulators for autoimmune- and allergic diseases. We explored several novel communication pathways between effector mechanisms of the immune system and certain nervous system functions, through the inflammatory cytokine network and steroid hormones. New methods and molecular constructs (e.g. antigen-targeting by engineered single-chain antibodies) and several modern confocal imaging techniques for studying cellular communication were also developed with the support of the grant

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins.

Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Posttranslational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C beta or with a rapamycin-inducible system in which various PI phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescent energy transfer (BRET)-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2, but not PtdIns(3,4,5)P3, was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasine, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]-Leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras mediated signals

Some new faces of membrane microdomains: A complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells

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 dil 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 dil 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. (c) 2007 International Society for Analytical Cytology

Sex hormone-binding globulin provides a novel entry pathway for estradiol and influences subsequent signaling in lymphocytes via membrane receptor

The complex effects of estradiol on non-reproductive tissues/cells, including lymphoid tissues and immunocytes, have increasingly been explored. However, the role of sex hormone binding globulin (SHBG) in the regulation of these genomic and non-genomic actions of estradiol is controversial. Moreover, the expression of SHBG and its internalization by potential receptors, as well as the influence of SHBG on estradiol uptake and signaling in lymphocytes has remained unexplored. Here, we found that human and mouse T cells expressed SHBG intrinsically. In addition, B lymphoid cell lines as well as both primary B and T lymphocytes bound and internalized external SHBG, and the amount of plasma membrane-bound SHBG decreased in B cells of pregnant compared to non-pregnant women. As potential mediators of this process, SHBG receptor candidates expressed by lymphocytes were identified in silico, including estrogen receptor (ER) alpha. Furthermore, cell surface-bound SHBG was detected in close proximity to membrane ERs while highly colocalizing with lipid rafts. The SHBG-membrane ER interaction was found functional since SHBG promoted estradiol uptake by lymphocytes and subsequently influenced Erk1/2 phosphorylation. In conclusion, the SHBG-SHBG receptor-membrane ER complex participates in the rapid estradiol signaling in lymphocytes, and this pathway may be altered in B cells in pregnant women

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

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

Differential expression of GL7 activation antigen on bone marrow B cell subpopulations and peripheral B cells

GL7 was originally described as a 35-kDa late activation antigen on mouse T and B cells. GL7 expression has also been demonstrated on thymocytes, germinal center B cells and some neuronal cell types. Flow-cytometry and immunohistochemistry were used to follow changes in the expression of GL7 during B cell development, amongst B cell subpopulations and various anatomical locations. GL7 is expressed as early as the pro-B cell stage and increases up to the pre-B-I stadium. Expression remains high on pre-B-II and on immature B cells, although slightly decreases during maturation. GL7 is almost completely downregulated when IgD appears on the cell surface. On the periphery only a few B cells are positive and these cells are almost exclusively found in the sIgD− germinal center areas of lymph nodes and spleen. The staining pattern of GL7 is very similar to that of PNA in the lymph nodes but in the bone marrow we have found both B220+PNA+GL7− and B220+PNA+GL7+ populations, showing that GL7 and the antigen recognized by PNA are different. After in vitro stimulation, the GL7hi B cell population has also been found to be IgD negative. Functional comparison between in vitro activated and MACS sorted GL7hi and GL7lo/− spleen B cells of immunized mice showed significantly higher specific and total antibody production as well as antigen presenting capacity in the GL7hi population