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Biomedical subjects

Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 109 records · Page 6Linked to original sources
PubMed1984

A selective defect in arachidonic acid release from macrophage membranes in high potassium media.

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.

Animals↗
PubMed1984

Effect of treatment on the cellular composition of cutaneous lesions in leprosy patients.

We report on the cellular composition of the cutaneous lesions of 38 leprosy patients prior to and after 12-18 months of chemotherapy. All of the patients with tuberculoid (TT) disease had OKT4/OKT8 ratios that exceeded 1.0. In the indeterminate patients, the ratio varied depending upon the immune status of the individual. A low ratio of OKT4/OKT8 cells (0.25) was found in the LL patients as previously reported. The low ratio of OKT4/OKT8 in the lesions of lepromatous patients did not undergo significant changes after treatment in spite of a marked reduction in the number of intracellular bacilli.

Humans↗
PubMed1983

The cutaneous infiltrates of leprosy. A transmission electron microscopy study.

The dermal lesions of 18 patients with leprosy have been examined by transmission electron microscopy. The patients exhibited a spectrum of disease from polar lepromatous to polar tuberculoid with intermediate stages in various states of therapy and relapse. The nature and quantities of inflammatory cells and bacteria have been determined by electron microscopy to supplement previous light and fluorescence microscopy studies. Lepromatous leprosy was characterized by many parasitized foam cells containing large, multibacillary vacuoles with intact, osmiophilic Mycobacterium leprae: Bacteria were embedded in an electron-lucent matrix. No extracellular bacteria were evident. Only small numbers of scattered lymphocytes were found. As one approached the borderline state, smaller numbers of bacilli were present as singlets and doublets in small vacuoles of macrophages. The more reactive forms showed increasing bacillary fragmentation, larger numbers of lymphoid cells, and an occasional epithelioid cell. At the tuberculoid end of the spectrum, clear evidence of an exuberant lymphocyte response was evident. Large numbers of T cells with extremely long and complex filipodia were closely associated with epithelioid and multinucleated giant cells. Many of the mononuclear phagocytes appeared nonviable, and areas of necrosis were evident. Bacillary remnants were scarce and the cytoplasm of the epithelioid cells contained occasional dense bodies and many stacks of endoplasmic reticulum and mitochondria. These results suggest that Leu 3a/OKT4 helper cells may be capable of driving the effector function of mononuclear phagocytes. This would lead to a significant microbicidal effect on M. leprae, perhaps through the production of toxic oxygen intermediates.

Humans↗
PubMed1983

Arachidonic acid metabolism by human monocytes. Studies with platelet-depleted cultures.

Purified human monocytes release and metabolize endogenous arachidonic acid (20:4) from phospholipid stores when challenged with particulate inflammatory stimuli or the calcium ionophore A23187. Using radiolabeled cultures, the percentage of total [3H]20:4 released was similar with each type of stimulus. However, the spectrum of 20:4 metabolites differed. With opsonized zymosan (OpZ) or Sephadex beads coated with IgG immune complexes (Ig-beads), the predominant product was thromboxane (25% of the total) together with smaller amounts of other cyclo-oxygenase products and lipoxygenase metabolites. Levels of thromboxane synthesis by monocytes were comparable to those by platelets, as measured by radioimmunoassay. In contrast, exposure to the nonspecific agent A23187 led to mainly lipoxygenase products (70% of the total). Monocytes isolated from mononuclear cell fractions of peripheral blood contain platelets specifically rosetted to their surfaces. These platelet contaminants were removed by sequential incubations of monocytes in serum and EDTA followed by adherence and detachment from tissue culture vessels. The presence of platelets in routinely isolated monocytes presented a major difficulty in the study of human monocyte 20:4 metabolism since platelets also synthesize thromboxane. Loss of 12-HETE synthesis (16-fold reduction relative to 5-HETE) in A23187-stimulated cultures provided a convenient measure of platelet depletion. This together with the response to monocyte-specific stimuli (OpZ and Ig-beads) allowed for the distinction between monocyte and platelet 20:4 metabolism.

Arachidonic Acid↗
PubMed1983

Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, influenza, vesicular stomatitis, and Semliki Forest viruses.

Exposure of a planar lipid bilayer to Sendai virus at pH 7.0 resulted in conductance increases that continued over several minutes, provided that the virus particles had first been conditioned by freezing and thawing, sonicating, or storing for 2 weeks in the cold. Individual electrical events could not be resolved, even on a millisecond time scale, and thus do not reflect the insertion of structural channels into the lipid bilayer. Prior treatment of the Sendai virions with protease prevented the conductance increases, but exposure of the bilayer to protease after induction of the conductance change did not abolish it. The Sendai-induced conductance change was increased in rate, but qualitatively unchanged in nature, if gangliosides were included in the planar bilayer. Activity for Sendai virus was low at pH 5.0, and increased with increasing pH up to 9.0. Influenza, Semliki Forest virus, and vesicular stomatitis virus all induced similar conductance changes around pH 5.2, but were inactive when tested at pH 7.0. The presence of cholesterol in the bilayer caused marked enhancement (two- to sixfold) of the response to Sendai, influenza and Semliki Forest virus, but caused only slight enhancement of the response to vesicular stomatitis virus. It is concluded that the observed increases in ionic permeability arise from alterations in lipid motions on a submillisecond time scale resulting from the incorporation of damaged viral membranes into the planar bilayer by fusion.

Cholesterol↗
PubMed1983

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

Animals↗
PubMed1983

Properties of the major outer membrane protein from Neisseria gonorrhoeae incorporated into model lipid membranes.

The major outer membrane protein from Neisseria gonorrhoeae was incorporated into artificial planar bilayer membranes by a detergent-dilution procedure. The integrated protein forms voltage-dependent aqueous pores with a minimal pore diameter estimated to be 11 A. A pore of this size suggests a role for this protein in macromolecular sieving at the level of the outer membrane. This protein self-associates preferentially in triplets of three equal unit conductance steps of 130 pS (in 0.1 M NaCl) each. The two-state model may be applied to explain the voltage-dependent conductance. The average lifetime of the open state of single channels is strongly dependent on the applied voltage, the channels shifting to the closed state at higher voltages. The pore is anion selective, differing from porins of other Gram-negative bacteria studied so far but resembling the voltage-dependent anion-selective channel of the outer membrane of mitochondria.

Bacterial Proteins↗
PubMed1983

Leukotriene C promotes prostacyclin synthesis by human endothelial cells.

Cultured endothelial cells from human umbilical vein were labelled with [3H]arachidonic acid for 16 hr. The radiolabel was localized primarily in phospholipids (93%) and 73% was distributed equally between phosphatidylcholine and phosphatidylethanolamine. Leukotriene C (10-1,000 nM) promoted a dose-dependent release of radiolabel into the culture medium. This response was 3.3 times control values at 100 nM. The major arachidonic acid metabolite synthesized was prostacyclin, which was 33% of the total released radiolabel. Endothelial cells also released small amounts of prostaglandin F2 alpha (6.1%), unidentified lipoxygenase products (14.8%), and unreacted arachidonic acid (33%). The 30-min time course of release was independent of the leukotriene C concentration used. Leukotriene D at similar concentrations also promoted endothelial cells to release primarily prostacyclin and unreacted arachidonic acid. The release of prostacyclin, a potent vasodilator agent, may be an important mediator in slow reacting substance effects on the vasculature.

Arachidonic Acid↗
PubMed1983

Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.

We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in delta psi, but a sustained depolarization lasting approximately equal to 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+,K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

Animals↗
PubMed1983

Mouse macrophage Fc receptor for IgG gamma 2b/gamma 1 in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore.

We tested the effect of specific ligands to the mouse macrophage IgG gamma 2b/gamma 1 Fc fragment receptor (FcR) on ion permeability of plasma membrane vesicles prepared from J774 macrophages by nitrogen cavitation. The monoclonal antibody directed against IgG gamma 2b/gamma 1 FcR (gamma 2b/gamma 1 FcR), 2.4G2 IgG, and soluble and immobilized immunocomplexes induces a dramatic cation flow through plasma membrane vesicles, as measured by [3H]tetraphenylphosphonium+ accumulation. Challenge with the monovalent 2.4G2 Fab also produces an ion flow but the effect is smaller by a factor of 2, and three other monoclonal antibodies directed against major surface antigens of mouse macrophages produce no net ion flow. Membrane vesicles incubated with FcR ligands do not discriminate between Na+ and K+ and show low permeability to Ca2+. gamma 2b/gamma 1 FcR was purified by using monoclonal 2.4G2 and reconstituted into proteoliposomes. Under these circumstances, the purified receptor increased the cation permeability of the proteoliposomes in the presence of specific ligands. The data indicate that the gamma 2b/gamma 1 FcR of J774 macrophages functions as a ligand-dependent ionophore. The ion influx into macrophages mediated by the FcR may play an important role as a signal for internalization of membranes and stimulus-secretion coupling.

Animals↗
PubMed1983

Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages.

In previous publications (Muller, W.A., R.M. Steinman, Z.A. Cohn. 1980, J.Cell Biol. 86:292-314), we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells, The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na(125)I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). (a) The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. (b) Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. (c) Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37 degrees C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2 degrees C and 22 degrees C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides.

Animals↗
PubMed1983

Internalization and degradation of macrophage Fc receptors during receptor-mediated phagocytosis.

Macrophage receptors for the Fc domain of immunoglobulin G (IgG) can mediate the efficient binding and phagocytosis of IgG-coated particles. After internalization, phagocytic vacuoles fuse with lysosomes, initiating the degradation of their contents. Using specific monoclonal and polyclonal antireceptor antibodies, we have now analyzed the internalization and fate of Fc receptors during the uptake of IgG-coated erythrocytes and erythrocyte ghosts by mouse peritoneal macrophages. Receptor-mediated phagocytosis led to the selective and largely irreversible removal of Fc receptors (greater than 50%) from the macrophage plasma membrane. The expression of several other plasma membrane proteins (including a receptor for complement), recognized by a series of antimacrophage monoclonal antibodies, was affected only slightly. Interiorized Fc receptors were rapidly and selectively degraded. This was demonstrated by a series of turnover studies in which Fc receptor was immunoprecipitated from lysates of 125I-labeled macrophages. These experiments were made possible by the development of a polyclonal rabbit antiserum, raised against isolated Fc receptor, which recognized the receptor even in the presence of bound ligand. In control cells, the receptor turned over with a t1/2 of approximately 10 h; after phagocytosis, greater than 50% of the receptors were degraded with a t1/2 of less than 2 h. The turnover of other unrelated plasma membrane proteins was unaffected (t1/2 of 18-23 h) under these conditions.

Animals↗
PubMed1983

Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.

GSH plays an important role in cellular defense against a wide variety of toxic electrophiles via the formation of thioether conjugates. We studied the role of GSH in murine tumor cell defense against a novel class of sulfhydryl-reactive antineoplastics, the sesquiterpene lactones (SL). Incubation of P815 mastocytoma cells with any of the four SL tested (vernolepin, helenalin, elephantopin, and eriofertopin) for 1 h resulted in 70-97% depletion of GSH. The importance of GSH resynthesis upon exposure of tumor cells to SL was evaluated with the use of buthionine sulfoximine (BSO), a selective, nontoxic inhibitor of gamma-glutamylcysteine synthetase. Inhibition of GSH synthesis with 0.2 mM BSO markedly enhanced SL-mediated cytolysis of four murine tumor cell lines. A 6- to 34-fold reduction in the amount of SL causing 50% lysis was obtained with BSO. Addition of BSO to P815cells either during or immediately after a 1-h pulse with 10 micrograms/ml of vernolepin increased cytolysis from less than 3% to 78-82%. However, a 1.5-h delay in the addition of BSO to such cells, which allowed for substantial resynthesis of GSH, reduced cytolysis to 30%. Recovery of GSH synthetic capacity after BSO treatment correlated with loss of the synergistic effect of BSO on lysis by vernolepin. BSO did not augment cytolysis by six other antineoplastics (doxorubicin, mitomycin C, vinblastine, cytosine arabinoside, maytansine, and 1,3-bis-[2-chloroethyl]-1-nitrosourea [BCNU]). Of these, only BCNU depleted cellular GSH. Lysis by jatrophone, another GSH-depleting antitumor agent, was increased 21-fold by BSO. Since prolonged incubation with BSO alone results in near-complete GSH depletion without loss of cell viability, SL-mediated cytolysis is probably not a result of GSH depletion. We have demonstrated, however, a critical role for GSH synthetic capacity as a determinant of tumor cell susceptibility to cytolysis by SL. GSH also plays an important role in cellular defense against oxidative injury. Vernolepin, acting as a GSH-depleting agent, markedly sensitized tumor cells to lysis by H2O2 (greater than 6.5-fold increase with 20 micrograms/ml of vernolepin). These findings suggest the possibility that the coordinated deployment of sulfhydryl-reactive antitumor agents, BSO, and oxidative injury might constitute an effective chemotherapeutic strategy.

Animals↗
PubMed1983

Leukotriene C release by macrophages.

Leukotriene C (LTC) and its metabolites leukotriene D and leukotriene E collectively make up the biological activity known as slow-reacting substance of anaphylaxis. Murine macrophages are potent sources of LTC (5(S)-hydroxy-6(R)-gamma-glutamylcysteinylglycyl-7,9-trans-11,14-cis-eicosatetr aenoic acid). Peritoneal and pulmonary tissue macrophages synthesize LTC and other arachidonic acid (20:4) metabolites in response to inflammatory stimuli such as unopsonized zymosan and IgG immune complexes. Peritoneal macrophages, in addition, release 20:4 when challenged with IgE immune complexes. These results suggest that macrophages may be a major source of leukotrienes in acute inflammation and also in immediate-type hypersensitivity reactions.

Animals↗
PubMed1982

The cutaneous infiltrates of leprosy: cellular characteristics and the predominant T-cell phenotypes.

We report on the characteristics of cells in the cutaneous lesions and blood of 21 patients with lepromatous, tuberculoid, and intermediate forms of leprosy. A large proportion of the infiltrates in lepromatous lesions consist of macrophages heavily parasitized with Mycobacterium leprae. The T cells in the lesions are devoid of OKT4/Leu 3a-positive ("helper") cells and consist almost exclusively of OKT8/Leu 2a-positive ("suppressor") populations. In contrast, the tuberculoid infiltrates contain well-organized epithelioid and giant-cell granulomas and only remnants of bacilli, and the predominant T cell is from the OKT4/Leu 3a-positive subset. In both tuberculoid and lepromatous infiltrates, T cells and macrophages expressed HLA-DR antigen. No marked alteration in the distribution of blood T-cell phenotypes was noted. We conclude that there is a marked difference between T-cell subsets in lepromatous and tuberculoid infiltrates, which may influence the microbicidal activity of macrophages in the lesions.

Antibodies, Monoclonal↗