Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen

A mathematical expression of the accumulation of the plasma cells in the spleen of CBA mice immunized intraperitoneally is presented. The dependence of the plasma cell reaction in the spleen on the kinetics of antigen concentration in the blood was confirmed. For the transition from antigen to plasma cells, index A was proposed. The mean values of index A were used for comparison of the calculated and experimental values of the plasma cell reaction and the recorded differences were not great. In a similar way, index A was used for prediction of plasma cell accumulation in the spleen of animals, immunized with a mixture of two soluble antigens — capsular antigen ofPasteurella pestis and complete antigen ofFrancisella tularensis. The calculated values of plasma cell reaction corresponded to experimental values.     

Plasma membrane lipid order and composition during adipocyte differentiation of 3T3F442A cells. Studies in intact cells with 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene

The plasma membrane lipid order of 3T3F442A cells was examined during the course of adipocyte differentiation by measuring the fluorescence polarization of 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene. This cationic fluorophore labels the plasma membrane but does not rapidly redistribute to intracellular organellar membranes and can, therefore, be used to specifically probe the plasma membrane of intact cells. Studies with whole cells demonstrated that the plasma membrane of 3T3F442A cells becomes less ordered during the course of adipocyte conversion and that this alteration begins relatively early during the differentiation process. In addition, the lipid order of plasma membranes isolated from adipocyte-stage cells was found to be lower than the lipid order of the early, fibroblast-stage cells. Analysis of membrane lipid composition suggests that the molecular bases for the decrease in adipocyte plasma membrane lipid order are a large increase in the level of monounsaturated phospholipid acyl chains and a decrease in the molar ratio of cholesterol to phospholipid. The alteration in plasma membrane lipid composition may be specifically required for integral membrane protein function, since the differentiation-dependent fatty acid desaturase activity is known to be maintained even in the absence of triacylglycerol accumulation.     

T11: a new protein marker on activated murine T lymphocytes

Murine lymphocytes were activated in vitro in mixed lymphocyte cultures (MLC) or by the addition of the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), or E. coli lipopolysaccharide (LPS). Activated lymphocytes were internally labeled with 35S-methionine and then disrupted by hypotonic lysis. A plasma membrane-enriched fraction was isolated from each cell population, and the 35S-labeled proteins in this fraction were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). An intensely labeled band, the position of which indicated an apparent m.w. of 11,000, was observed when plasma membrane-enriched fractions from MLC- and Con A-activated cells were subjected to SDS-PAGE. In contrast, plasma membrane-enriched fractions from normal spleen cells, LPS-activated cells, PHA-activated cells, and EL4, RDM4, and P815 tumor cells possessed little or none of this protein, which we have designated T11. T11 was not found in the soluble cytoplasmic protein from MLC-activated cells. Hence the presence of T11 in the plasma membrane-enriched fraction from these cells cannot be attributed to contamination by cytoplasmic protein. Removal of T cells from populations of MLC-activated cells by treatment with monoclonal anti-Thy 1 and complement removed T11. These results suggest that T11 may represent a new protein marker on a subclass of activated T lymphocytes.     

Studies on the activation mechanism of human mononuclear leukocytes isolated by physical methods

Responder lymphocytes, accessory lymphocytes and monocytes were isolated using countercurrent centrifugal elutriation and free flow electrophoresis. The Concanavalin A (Con A) binding behaviour of the isolated cell populations was studied. Responder lymphocytes and accessory cells bound Con A by different mechanisms. Cells in all the isolated populations were able to interact directly with Con A. Con A binding to responder lymphocytes was inhibited by alpha-methylmannoside (alpha MM) and by a distinct plasma protein. Accessory lymphocytes and monocytes bound Con A even in the presence of 10% human plasma or 50 mM alpha MM. The plasma protein which inhibited interaction of responder lymphocytes with Con A also reduced lymphocyte proliferation, when resting monocytes were used as accessory cells. If, however, monocytes were used after activation by lipopolysaccharide no inhibition effect was observed. From the results we conclude that there is a distinct plasma protein which protects the organism against lymphocyte stimulation not controlled by accessory cells. This inhibitory plasma protein appears to be a euglobulin.     

Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.     

Defective glycoproteins in the plasma membrane of an aggregation minus mutant of Dictyostelium discoideum uith abnormal cellular interactions

Experiments involving the co-incubation of wild type (A3) cells of Dictyostelium discoideum and a spontaneous aggregation-minus mutant (HW 2) suggested that the mutant was defective in cellular interactions. The inhibition of A3 development by HW 2 cells and the differentiation of a small fraction of HW 2 cells which is allowed by A3 cells, both depend on cell contact. Therefore, we compared cell surface molecules in vegetative A3 and HW 2 cells by a variety of techniques to determine whether defects in HW 2 could be found prior to the inhibition of development in vegetative amoebae. Antigenic defects, or differences in binding of concanavalin A, or both, were localized to three plasma membrane macromolecules using glutaraldehyde-fixed sodium dodecyl sulfate gels of plasma membranes. Two periodic acid-Schiff-positive glycoproteins, and one glycolipid also differed in HW 2. Three glycoproteins had an increased sensitivity to pronase in isolated plasma membranes suggesting an alteration in their topography. Glycoprotein E, the major glycoprotein of vegetative plasma membranes is abnormal in topography, altered as a concanavalin A receptor, and is antigenically abnormal.     

A monoclonal antibody with reactivity restricted to normal and neoplastic plasma cells

A monoclonal antibody that defines a new and distinct plasma cell antigen, termed PC-1, was developed against human plasmacytoma cells. This antigen is strongly expressed on normal plasma cells isolated from bone marrow and on abnormal plasma cells isolated from myelomas, plasma cell leukemias, and plasmacytomas. The antigen is not detected on normal T or B lymphocytes, granulocytes, or monocytes, and with the exception of plasma cells, is absent on malignancies of B, T, or myeloid origin. Utilizing pokeweed mitogen to induce human B lymphocyte differentiation in vitro, PC-1 is expressed when B cell determinants are lost and the plasmacytoid morphology, intracytoplasmic immunoglobulin-staining, and surface PCA-1- and T10-staining characteristic of plasma cells appear. This antigen is useful for the study of the terminal stages of normal B cell differentiation to plasma cells, and may offer insight into the heterogeneity of the plasma cell dyscrasias.     

Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.     

Localization of a putative cell adhesion molecule (gp110) in Wistar and Fischer rat tissues

A plasma membrane glycoprotein (gp110) involved in cellular adhesion was studied in Wistar and Fischer rats. For quantitative analysis of the gp110 molecule a sandwich-ELISA was used. High quantities of gp110 were found especially in the liver, small intestine, submandibular gland and lung. The distribution and localization of the gp110 were investigated by immunohistochemistry utilizing soluble complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase antibodies. Immunoreactivity was present in plasma membranes of vascular endothelial cells of some organs. Furthermore, immunostaining also occurred in plasma membranes of lymphocytes, exocrine gland cells, excretory duct cells, hepatocytes, epithelial cells of the small intestine, kidney and vesicular gland and in the cytoplasm of renal connecting and collecting duct cells. The localization of gp110 in the luminal domain of the plasma membrane at many sites suggests that this glycoprotein is also involved in processes distinct from cell adhesion.     

Surface analysis and depth profiles of calcium in hepatoma cells during pyruvate-induced DNA synthesis

Induction of DNA synthesis is associated with increased uptake of calcium in cultured cells. Calcium distribution within the plasma membrane and adjacent cytoplasmic layers of hepatoma cells was investigated with X-ray photoelectron spectroscopy and oxygen plasma etching. Cells in minimal growth medium initiate active DNA synthesis 16 h after addition of sodium pyruvate. Cells stimulated with pyruvate and pyruvate-free controls were analysed by X-ray photoelectron spectroscopy--oxygen plasma etching at 0--40 A (layer I), 0--450 A (layer II) and 0--4000 A (layer III from the outer cell surface. Calcium concentrations were elevated in induced cells compared with controls: +20% in layer I, +60% in layer II, and +300% in layer III. As the plasma membrane is 90--120 A thick, these results indicate that pyruvate-induced DNA synthesis is preceded by an increase in calcium, most marked in the cytoplasm subjacent to the plasma membrane, moderate at its inner surface, and minimal at its outer surface.     

Effects of oestradiol-17 beta and progesterone on the number of plasma cells in uteri of ovariectomized mice

Immunoglobulins A and G were localized by immunoperoxidase labelling in uteri of ovariectomized mice treated with oestradiol-17 beta and progesterone. The administration of oestradiol or progesterone alone to ovariectomized mice for 3 days increased the number of IgA plasma cells from about 1 to 14 per histological section. When the two hormones were administered simultaneously for 3 days the number of plasma cells per section was equal to or greater than with either hormone alone. Treatment with oestradiol followed by progesterone in a sequence that prepares the uterus for implantation resulted in about 31 IgA plasma cells per section. Counts of IgG plasma cells showed similar trends but the numbers were smaller. The results indicate that progesterone increases rather than decreases the number of plasma cells in the mouse uterus. This is consistent with observations on intact mice during oestrus and pregnancy and suggests that the marked increase in endometrial plasma cells at the time of implantation in mice is a response to progesterone acting on an oestrogen-primed uterus.     

Chronic ethanol increases liver plasma membrane fluidity

Purified plasma membrane fractions of cultured well-differentiated Reuber H35 hepatoma cells were studied after growth in the presence or absence of ethanol. Growth of cells in the presence of ethanol significantly increased plasma membrane 5'-nucleotidase activity but did not influence sodium-potassium adenosinetriphosphatase activity. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from cells grown in 80 mM ethanol for 3 weeks, compared to controls. Decreased polarization of DPH in plasma membranes was observed after 3-weeks growth of cells in as little as 1 mM ethanol. A 1-h exposure to 80 mM ethanol had no effect. Altered DPH polarization was due to a decrease in the order parameter of the probe. The rotational correlation time of the probe was virtually unchanged. Chronic ethanol treatment of cells did not alter the polarization of the membrane surface probe trimethylammoniodiphenylhexatriene. Plasma membranes from cells grown in 80 mM ethanol had decreased contents of both phospholipid and unesterified cholesterol, but the cholesterol to phospholipid ratio was unchanged. The percentages of sphingomyelin and phosphatidylserine in plasma membrane phospholipids were significantly decreased after ethanol treatment, while the phosphatidylcholine/sphingomyelin ratio was increased by 42%. Vesicles prepared from total plasma membrane lipids of ethanol-treated cells, as well as vesicles prepared from polar lipids alone, showed the same alterations in DPH polarization as did plasma membranes. The importance of ethanol metabolism in the observed plasma membrane changes was demonstrated in two ways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Internalized Plasma Membrane Cholesterol Passes through an Endosome Compartment That Is Distinct from the Acid Vesicle–Lysosome Compartment

Cholesterol from the plasma membrane of MA-10 Leydig tumor cells is internalized into the cell and either esterified or used as substrate for steroid hormone synthesis. In the present studies we show that chloroquine and sphinganine cause LDL cholesterol and cholesteryl esters to accumulate in the cells. A lysosome fraction contained the excess cholesterol and cholesteryl esters. Both inhibitors blocked the conversion of plasma membrane cholesterol into intracellular cholesteryl esters and caused dose-dependent inhibition of dibutyryl-cAMP-stimulated progesterone synthesis. Radiolabeled cholesterol applied to the plasma membrane of MA-10 cells accumulated in the lysosome fraction of chloroquine and sphinganine-treated cells. Evidence that these inhibitors did not require the Golgi was provided by experiments using brefeldin A. Experiments utilizing a fluorescent cholesterol analogue and a lysosomal marker indicated that cholesterol entered the cells in structures that were different than the acidic vesicle–lysosome compartment. Consistent with this observation was the observation that the peak fluorescence fractions of cells subjected to density gradient centrifugation was of lower density than the lysosome fraction.     

Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.     

Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission

Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.     

Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells.

Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.