Cell Membrane Molecules Research Articles

Effects of copper and zinc on chondrocyte-mononuclear cell adhesion via ICAM-1/CD18 interactions

Copper and zinc play important, but as yet undetermined, roles in the function of the immune system and in inflammatory processes. In this study we demonstrated that addition of copper salts to cultures of rat and human articular cartilage and isolated porcine chondrocytes induced expression of the intercellular adhesion molecule, ICAM-1. Expression of this adhesion molecule in response to copper sulphate did not, however, promote adhesion of chondrocytes to peripheral blood mononuclear cells (PBMC), whereas ICAM-1 induced by IL-lα significantly increased cell adhesion. Zinc sulphate, in contrast to copper sulphate, had no effect on ICAM-1 expression on chondrocytes, but was found to upregulate CD18 (LFA-1), the counter-receptor for ICAM-1 on PBMC, thereby increasing the adhesion of PBMC to chondrocytes. The possible consequences of these novel effects of copper and zinc salts on cell membrane molecules are discussed.

The purinergic P2Z receptor of human macrophage cells. Characterization and possible physiological role.

We have investigated responses of human monocyte/macrophage cells to extracellular ATP (ATPe). Freshly isolated peripheral blood monocytes showed responses linked to P2Y but not P2Z purinergic receptors; however, during in vitro macrophage differentiation, these cells also exhibited responses suggestive of the presence of the membrane-permeabilizing P2Z receptor. In fact, in human macrophages a brief (15-min) exposure to ATPe, but not other nucleotides, caused (1) a rapid and long-lasting plasma membrane depolarization; (2) a large increase in intracellular Ca2+ concentration followed by efflux of the Ca2+ indicator; (3) uptake of low molecular weight hydrophilic molecules such as Lucifer yellow and ethidium bromide; and (4) cell rounding, swelling, and eventual release of the cytoplasmic enzyme lactate dehydrogenase. rIFN-gamma enhanced both membrane-permeabilizing and cytotoxic ATPe effects. Membrane permeabilization and cytotoxicity were fully blocked by pretreatment of the cells with oxidized ATP, a compound recently shown to block P2Z receptors covalently in macrophages. Blocking of the P2Z receptor by oxidized ATP also inhibited multinucleated giant cell generation stimulated by concanavalin A or rIFN-gamma without decreasing monocyte migration or membrane adhesion molecule expression. These data suggest that human macrophages express rIFN-gamma-modulated purinergic P2Z receptors in vitro and hint at a role for these plasma membrane molecules in the generation of macrophage polykarions.

T cells which do not express membrane tumor necrosis factor-alpha activate macrophage effector function by cell contact-dependent signaling of macrophage tumor necrosis factor-alpha production.

Previous studies have suggested that T cell contact-dependent signaling of macrophages (Mphi) is mediated by membrane tumor necrosis factor-alpha (memTNF-alpha), based on the observation that anti-TNF-alpha could inhibit T cell-mediated Mphi activation. The current report confirms that anti-TNF-alpha does inhibit activation of interferon-gamma (IFN-gamma)-primed Mphi by paraformaldehyde-fixed activated T cells. However, the involvement of membrane molecules other than memTNF-alpha in the contact-dependent signaling is suggested by two lines of evidence. First, the TH2 clone, AK8, displayed neither secreted TNF-alpha/beta nor memTNF-alpha/beta detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact-dependent signaling of M phi activation as TH2 and TH1 cells which do express memTNF-alpha. Second, the expression of memTNF-alpha by the TH2 clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate Mphi is maximal at 6-8 of activation and declines thereafter. Since TNF-alpha is known to play a critical role in activation of Mphi effector function, it was hypothesized that T cell membrane components other than memTNF-alpha might signal Mphi production of TNF-alpha, thus allowing autocrine TNF-alpha stimulation of Mphi effector function. In support of this, it is demonstrated that paraformaldehyde-fixed activated TH2 cells can induce de novo production and release of TNF-alpha by Mphi. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde-fixed resting T cells did not induce TNF-alpha gene expression. Previous studies have demonstrated a role for autocrine TNF-alpha stimulation in LPS induction of effector function in recombinant IFN-gamma-primed Mphi. The current study confirms that TNF-alpha plays a critical role in T cell contact-dependent signaling of Mphi but indicates that memTNF on the T cells may not be a sine qua non factor for contact-dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of Mphi effector function by stimulation of M phi TNF-alpha production.

Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions.

Cardiotoxins (CTXs) are a group of homologous proteins found in cobra snake venom and consist of 60-62 amino acid residues. Although CTXs are known to consist of three extended beta-sheet loops similar to neurotoxins, the target and interaction of CTXs with membranes unlike those of neurotoxins are not well understood. Herein, we report comparative studies of 10 CTXs purified from Taiwan cobra (Naja naja atra) and Mozambique spitting cobra (Naja mossambica mossambica) snake venoms with respect to their interactions with zwitterionic phospholipids. Based on the CTX-induced mixing of sphingomyelin vesicles and the binding of CTX to lysophosphatidylcholine micelles, two distinct types of CTX, i.e. P- and S-type CTX, are identified. P-type CTXs are characterized by the presence of Pro-31 within a putative phospholipid binding site near the tip of loop 2; whereas S-type CTXs are characterized by the presence of Ser-29 within the same but more hydrophilic region. Although binding of all CTXs to phospholipid membranes involves a phospholipid binding site at loop 1, P-type CTXs exhibit higher fusion and binding activity than S-type CTXs, presumably due to the additional phospholipid binding site at loop 2. The binding modes of P- and S-type CTX are thus different. Analysis of the primary structures of 46 CTXs from the genus Naja indicates that these two types of CTXs exist in all species examined. Reasonable structure/activity correlation can be detected for the effects of CTXs on muscle and red blood cells, although notable exceptions are also found. S-type CTXs are generally found to exhibit higher muscle cell depolarization activity, whereas P-type CTXs are found to possess a higher hemolytic activity. Thus the mechanism of action of CTXs seems to involve CTX-membrane interactions and depends on the type of the cell membrane and CTX molecules under study. The two lipid binding sites in P-type CTXs and one lipid binding site in S-type CTXs show large variation in their amino acid residues, but they do display some common distribution of residue type. Analogous to the signal sequences for protein import, these regions are characterized by the coexistence of an exposed hydrophobic surface flanked on either side by a cationic residue. A hypothesis is proposed to explain the general cytotoxic and specific cardiotoxic effect of CTXs based on the two CTX subtypes in snake venom.

Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) and Griffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha-D-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances has approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage. Removal of oxygen may allow excited state fluorophores in close proximity to the plasma membrane to react with neighboring proteins or lipids during photobleaching. This effect has important implications for the ability of the fluorescence photobleaching recovery technique to report accurate lateral mobilities of cell membrane molecules under hypoxic conditions.

Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.

Simian immunodeficiency virus (SIVmac251) membrane lipid mixing with human CD4+ and CD4- cell lines in vitro does not necessarily result in internalization of the viral core proteins and productive infection.

The cell binding site of simian immunodeficiency virus (SIV) is believed to be the CD4 molecule. Several CD4+ cell lines are, however, resistant to infection by SIVmac251 in vitro and additional cell membrane molecules have been implicated in SIVmac251 entry. We investigated the binding, envelope fusion and entry of the viral core proteins (p27) of SIVmac251 into two human CD4+ cell lines (H9 and Sup-T1) which are infectible, and one CD4+ (A3.01) and two CD4- cell lines (K562 and Raji) that are resistant to infection. The fusion of the viral and cellular membranes was monitored by a fluorescence assay for lipid mixing. Cell entry of the viral core was evaluated following virus-cell incubation and cell surface trypsinization. We found that SIVmac251 can bind to and fuse (membrane lipid mixing) in a temperature-dependent but pH-independent fashion with CD4+ and CD4- human-derived cell lines. In contrast, lipid mixing with CD4 expressing EL-4 mouse T cells or Mv-1-lu mink lung fibroblasts was absent or limited, suggesting that certain components of human cell membranes in addition to CD4 are involved in SIVmac envelope-cell fusion. Lipid mixing with the human cells was inhibited partially by soluble CD4. Anti-CD4 antibodies inhibited the lipid inter-mixing with H9, but not with Raji cells, whereas neutralizing anti-SIVmac sera inhibited fusion with both CD4+ and CD4- cells. Out of the five human cell lines tested, efficient entry of p27 and productive infection took place only with H9 and Sup-T1 cells. In these two cases, the amounts of p27 internalized during virus-cell fusion correlated with the extent of infection.

Electrochromic behaviour of carotenoid molecules in nerve cell membranes: A resonance Raman study

It was found that, following the depolarization of cell membranes of frog sciatic nerve, the 1521 and 1156 cm −1 resonance Raman (RR) bands of carotenoids showed an increase in intensity of 20%, 15% and 4% at excitation wavelengths of 488, 496.5 and 514.5 nm respectively. None of the bands revealed any change with excitation at 441.6, 457.9, 472.7 and 476.5 nm. These results can be explained by an electrochromic shift of the excitation profile of the RR bands by about 0.5 nm to the red. A dependence of the 1521 cm −1 band intensity on transmembrane potential was obtained. Under the effect of lanthanum ions, which are known to modify the surface charge of the membrane, the intensity of the Raman bands decreased by 150%–20% (excitation, 488.0 nm). The results obtained indicate that RR spectroscopy of carotenoids can be used to monitor changes in the transmembrane potential and surface charge of nerve cell membranes.

T cell-dependent B cell activation.

B cells obtain help from T cells in the antibody response by acting as antigen-specific antigen presenting cells. A direct signal through binding of antigen to membrane Ig can enhance B cell antigen presentation and T-dependent B cell activation, but is not required for a productive interaction between a small resting B cell and a differentiated helper T cell. As a result of helper T cell recognition of antigen on the B cell surface, the T cell becomes activated and in turn activates the B cell. T cell help has two components: lymphokines which act as growth and differentiation factors for B cells, and additional signals which require cell contact and enable B cells to respond to lymphokines. Contact help activity is regulated like lymphokine synthesis and secretion. Because contact help activity is retained by fixed, activated helper T cells and plasma membranes prepared from activated T cells, contact help is likely to be owing to new proteins expressed as membrane-bound lymphokines or activation antigens on helper T cells. Once induced, contact help can be delivered to B cells independently of recognition of antigen/class II MHC. A newly identified activation antigen of helper T cells, a ligand for the B cell differentiation antigen, CD40, is a key component of contact help. The roles of other T and B cell membrane molecules in contact help are reviewed.

Transcellular Intestinal Calcium Transport in Freshwater and Seawater Fish and its Dependence on Sodium/Calcium Exchange

ABSTRACT Transepithelial calcium uptake and transcellular calcium uptake mechanisms were compared in the proximal intestine of freshwater- and seawater-adapted tilapia, Oreochromis mossambicus. Stripped intestinal epithelium of seawater fish showed a higher paracellular permeability to calcium in vitro. Net transepithelial calcium uptake was 71% lower, reflecting a physiological response to the increased inward calcium gradient. Na+/K+-ATPase activity was significantly enhanced in enterocytes of seawater- adapted fish, in line with the water transport function of the intestine in seawater fish. The Vmax and the Km values for Ca2+of the ATP-dependent Ca2+ pump in seawater fish enterocytes were 28% and 27%, respectively, lower than in freshwater fish. The Km for Ca2+ of the Na+/Ca2+ exchanger was 22% lower, and a 57% decrease in the Vmax for Ca2+ of the exchanger was observed. Apparently, the density of exchanger molecules in the basolateral plasma membrane is reduced in seawater fish. From the correlation between the differences in net intestinal calcium uptake and Na+/Ca2+ exchange activity we conclude that Na+/Ca2+ exchange is the main basolateral effector of transcellular calcium uptake.

Dendritic cells have reduced cell surface membrane glycoproteins including CD43 determinants.

The molecular basis for the special ability of dendritic cells (DC) to stimulate primary T cell responses is at present unknown. Important accessory molecules such as the adhesion molecules LFA-3, LFA-1, ICAM-1 and ICAM-2 and probably BB1/B7 (the ligand for T cell CD28 and CTLA-4) are expressed on DC1 and contribute to the antigen presenting function of DC. However no unique co-stimulatory cell membrane molecule or secreted cytokine has been identified on DC to date. Human blood, tonsil and bone marrow DC are potent stimulators of alloimmune T lymphocyte responses. Highly purified peripheral blood CD19 positive B cells and CD14 positive monocytes may also stimulate an alloresponse from peripheral blood T cells, including those carefully depleted of endogenous antigen presenting cells; albeit a less potent one.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker.

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone.

Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.

In vitro sperm capacitation to treat antisperm antibodies bound to the sperm surface.

Our objective was to study antisperm antibody bound to the acrosome region during in vitro capacitation and to determine whether acrosome-antibody free sperm can be obtained from previously acrosome-antibody-coated sperm. The spermatozoa from a selected series of 14 patients were tested for sperm antibodies bound to the sperm surface using d-IBT and focusing on the acrosome positivity. The tests were carried out before the incubation and after 3, 6, 9, and 12 h of incubation in Tyrode's solution with 0.5% human serum albumin as the capacitation medium. Tests to evaluate acrosome region, sperm motion parameters, and zonae binding ability were carried out. In this way we were able to evaluate sperm function during capacitation protocol. The patients were 14 subjects selected according to good seminal characteristics, good post-rise sperm parameters, and high percentage of ASA bound to the sperm surface. In all cases the results showed that antisperm antibodies bound to the acrosome region were shed prior to the acrosome reaction. During sperm capacitation in human a modification, migration, or shedding of plasma membrane molecules takes place. The presence of antibodies in such an important area of the sperm head could certainly interfere in the fertilization process. Our data indicate that in vitro capacitation could provide an in vitro therapy capable of eluting antibodies from the acrosome region.

Dynamic Physical Interactions of Plasma Membrane Molecules Generate Cell Surface Patterns and Regulate Cell Activation Processes

Molecular interaction and transmembrane signal transducing events generate a very dynamic and ever changing «pattern» in the plasma membranes. Lymphocytes, the key functional elements of the immune system, are eminently suited to be the primary targets to investigate these proximity, mobility, or other physical-chemical changes in their plasma membranes. Recently, a number of experiments suggested that processed peptides from antigens can bind specific components of MHC molecules ( Elliott et al., 1991 ). This is certainly a way to alter their structure. Cell surface patterns of topological nature, assembly and disassembly of oligomeric receptor structure like the IL-2 receptor have been investigated by sophisticated biophysical techniques. The dynamic changes in the two-dimensional cell surface pattern and intramolecular conformational changes within this «larger» macro-pattern may have a strong regulatory role in signal transducing and intercellular recognition processes. Recent data on these problems are presented together with brief and critical discussions.

Cellular protein profiles of the Hodgkin's disease cell lines L428, KM-H2 and HDLM-2: A comparative study

The origin of the malignant mononuclear Hodgkin's cell and the classic Reed-Sternberg cell in Hodgkin's disease (HD) remains controversial despite extensive immunohistological and lymphoid gene analysis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with protein staining and radioactive labelling has not been fully exploited in analysing the HD-derived cell lines available. The NP40 solubilised cellular proteins from the three HD cell lines, L428, KM-H2 and HDLM-2 were analysed using these techniques and compared with other haemopoietic cell lines and leucocytes of myeloid and lymphoid origin. The electrophoretic patterns of the three HD cell lines, although clearly different from one another, had many features in common. No major differences between the cell types were detected by Coomassie brilliant blue staining. The HD cell lines were more readily distinguished from the myeloid and to a lesser extent the lymphoid cell lines by silver staining, but HD cell line specific proteins (13, 19, 36, 60, 150 kD) were detected only on one line, L428. Iodination of cell membrane molecules, SDS-PAGE and subsequent autoradiography revealed three molecules (118, 22, 12 kD) which were restricted to the HD cell lines and the B-cell line Mann, and one molecule (144 kD) restricted to the HD cell lines and U937. Molecules unique to HDLM-2 (211 kD) and L428 (46 kD) were also detected by this method. Cell surface labelling with NaB 3H 4 identified a glycoprotein of 102 kD limited to HDLM-2 and L428, as well as a glycoprotein of 97 kD present on KM-H2 alone and one of 63 kD on L428 alone. Overall the HD cell line protein profiles displayed little similarity to the patterns of the other cell types studied and provide further evidence to support functional and phenotypic studies which identify Hodgkin's cells as a unique cell type. The molecules identified as HD cell line restricted may have potential as markers for this cell type.

Evidence for a membrane defect in Alzheimer disease brain.

To determine whether neurodegeneration in Alzheimer disease brain is associated with degradation of structural cell membrane molecules, we measured tissue levels of the major membrane phospholipids and their metabolites in three cortical areas from postmortem brains of Alzheimer disease patients and matched controls. Among phospholipids, there was a significant (P less than 0.05) decrease in phosphatidylcholine and phosphatidylethanolamine. There were significant (P less than 0.05) decreases in the initial phospholipid precursors choline and ethanolamine and increases in the phospholipid deacylation product glycerophosphocholine. The ratios of glycerophosphocholine to choline and glycerophosphoethanolamine to ethanolamine were significantly increased in all examined Alzheimer disease brain regions. The activity of the glycerophosphocholine-degrading enzyme glycerophosphocholine choline-phosphodiesterase was normal in Alzheimer disease brain. There was a near stoichiometric relationship between the decrease in phospholipids and the increase of phospholipid catabolites. These data are consistent with increased membrane phospholipid degradation in Alzheimer disease brain. Similar phospholipid abnormalities were not detected in brains of patients with Huntington disease, Parkinson disease, or Down syndrome. We conclude that the phospholipid abnormalities described here are not an epiphenomenon of neurodegeneration and that they may be specific for the pathomechanism of Alzheimer disease.

In vitro transcription faithfully reflecting T-cell activation requirements.

T-cell activation is a complex process mediated by cell membrane molecules including the T-cell antigen receptor (TCR), adhesive molecules, and cytokine receptors that collectively produce an increase in intracellular Ca2+, and activation of protein kinase C that initiate a genetic program resulting in immunologic function and irreversible differentiation. To understand how these cell membrane events are translated into a genetic regulatory cascade resulting in T-cell function, we have developed an in vitro transcription system, derived from Jurkat T-cells, which demonstrates inducible, cell-type-specific transcription following T-cell stimulation. Nuclear extracts from cells stimulated with phorbol 12-myristate 13-acetate and ionomycin, which activate protein kinase C and mimic physiological activation through the T-cell antigen receptor, transcribe an interleukin-2 (IL-2) enhancer (-326 to +24) template 5-fold more efficient than nuclear extracts from resting T-cells and severalfold more efficient than extracts from Jurkat cells treated with phorbol 12-myristate 13-acetate or ionomycin alone. Further results demonstrate that in vitro transcription of the IL-2 enhancer is T-cell specific since nuclear extracts from rat liver and stimulated HeLa cells are unable to induce IL-2 transcription. The activation-dependent, T-cell-specific in vitro transcription system described here should facilitate the dissection of signals that emanate from the T-cell surface resulting in IL-2 transcription and T-cell activation.

Five novel antigens illustrate shared phenotype between mouse thymic stromal cells, thymocytes, and peripheral lymphocytes

Previously we characterized by immunohistology a group of rat anti-mouse thymic stromal mAbs (MTS 12, 32, 33, 35, and 37), which recognized novel plasma membrane determinants on both thymic stromal cells (TSCs) and thymocytes. The present study investigates in more detail this incidence of shared phenotype by an extensive flow cytometric analysis of MTS mAb reactivity on TSCs, thymocyte subsets, peripheral lymphocytes, and bone marrow cells. Examination of freshly isolated or cultured heterogeneous TSCs and TSC clones confirmed that the mAb identified plasma membrane molecules on distinct subsets of these cells. All but MTS 12 reacted with epithelial cells. Triple-labelling illustrated that MTS 32, 33, and 37 were also reactive with more than 90% of total thymocytes, but varied in their distribution on the four major CD4 and CD8 defined subsets. MTS 12, staining with thymic vascular endothelium by immunohistology, labelled more than 95% of each subset. MTS 35 reactivity in each subset correlated strongly with only the immature populations. Examination of peripheral lymphocytes by triple- and double-labelling unexpectedly showed that MTS 33, 35, and 37 did not recognize peripheral T cells but labelled all B cells. MTS 32 was negative for B cells, but positive for all CD8+ T cells, yet only a subset of CD4+ T cells. Further, MTS 33, 35, and 37 were present on a significant percentage of bone marrow cells. MTS 12 reacted with virtually all peripheral T and B cells, and about 50% bone marrow leukocytes. Collectively these results reveal the same novel epitopes on different thymic cell types and subsets thereof, highlighting specific similarities between cells of apparently diverse lineages. These findings may be of importance in the delineation of intercellular communications within the thymus and emphasize the integrated nature of the microenvironment in this organ.