Sylvia Hoffstein

YIN/YANG MODULATION OF LYSOSOMAL ENZYME RELEASE FROM POLYMORPHONUCLEAR LEUKOCYTES BY CYCLIC NUCLEOTIDES*

Since 1970 it has been clear that polymorphonuclear leukocytes of man and other species selectively release lysosomal, but not cytoplasmic, constituents when exposed to particulate challenges.] :i Originally demonstrated after the uptake of zymosan particles,’, this phenomenon also includes the release of lysosomal constituents when white cells are exposed to immune complexes either in the bulk phase or dispersed upon The former process, release of lysosomal hydrolases during phagocytosis of discrete particles by white cells, has been termed “regurgitation during feeding,” whereas the latter, extrusion of lysosomal enzymes after the exposure of white cells to immune complexes on a supporting surface, has been termed “reverse endocytosis” 1 or “frustrated phagocytosis.” i . l1 In these two circumstances, the white cells release such enzymes as /

Thyrotropin-releasing Hormone Stimulation of Prolactin Release from Clonal Rat Pituitary Cells: EVIDENCE FOR ACTION INDEPENDENT OF EXTRACELLULAR CALCIUM

glucuronidase, acid phosphatase, aryl sulfatase, and myeloperoxidase (constituents of azurophil granules), in addition to lysozyme and alkaline phosphatase (constituents of specific granules) .2-11 Viability is maintained during release, and it has become appreciated that the stimuli to release of lysosomal hydrolases act via Fc receptors on the polymorphonuclear leukocytes that recognize altered immunoglobulins, such as those present in the form of immune complexes, and via C3 receptors that recognize C3b fragments present upon such particles as serum-treated zymosan.lo,

Introduction of enzymes, by means of liposomes, into non-phagocytic human cells in vitro

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and (45)Ca(2+) efflux from GH(3) cells, a clonal strain of rat pituitary cells. Elevation of extracellular K(+) also induces prolactin release and increases (45)Ca(2+) efflux from these cells. In this report, we distinguish between TRH and high K(+) as secretagogues and show that TRH-induced release of prolactin and (45)Ca(2+) is independent of the extracellular Ca(2+) concentration, but the effect of high K(+) on prolactin release and (45)Ca(2+) efflux is dependent on the concentration of Ca(2+) in the medium. The increment in (45)Ca(2+) efflux induced by 50 mM K(+) during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca(2+) from 1,500 to 0.02 muM (by adding EGTA), whereas 1 muM TRH enhanced (45)Ca(2+) efflux similarly over the entire range of extracellular Ca(2+) concentrations. Although 50 mM K(+) caused release of 150 ng prolactin from 40 x 10(6) GH(3) cells exposed to 1,500 muM Ca(2+) (control), reduction of extracellular Ca(2+) to 2.8 muM decreased prolactin release caused by high K(+) to <3% of controls and no prolactin release was detected after exposure to 50 mM K(+) in medium with 0.02 muM free Ca(2+). In contrast, TRH caused release of 64 ng of prolactin from 40 x 10(6) GH(3) cells exposed to medium with 1,500 muM Ca(2+), and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 muM Ca(2+), respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of (45)Ca(2+) from GH(3) cells in static incubations with 1,500 or 3.5 muM Ca(2+), hereby confirming that the enhanced (45)Ca(2+) efflux caused by TRH in both low and high Ca(2+) medium was not an artifact of the perifusion system.Data obtained with chlortetracycline (CTC), a probe of membrane-bound Ca(2+), were concordant with those obtained by measuring (45)Ca(2+) efflux. Cellular fluorescence of CTC varied with the extracellular Ca(2+) concentration and the duration of incubation. TRH decreased the fluorescence of cell-associated CTC in a manner strongly suggesting stimulus-induced mobilization of Ca(2+), and this effect was still demonstrable in GH(3) cells incubated in 50 mM K(+). These data suggest that TRH acts to mobilize sequestered cell-associated Ca(2+) reflected as a (45)Ca(2+) efflux which is independent of the extracellular Ca(2+) concentration. Mobilization of sequestered Ca(2+) into the cytoplasm may elevate free intracellular Ca(2+) and serve to couple stimulation by TRH to secretion of prolactin.

Mechanisms of lysosomal enzyme release from human leucocytes: III. Quantitative morphologic evidence for an effect of cyclic nucleotides and colchicine on degranulation

Abstract Liposomes containing entrapped horsedish peroxidase were incubated with three human cell lines in vitro. Although these cells did not ingest latex particles, and took up less than 1 minut of free peroxidase/5 · 106 from the medium, significant amounts (41–164 munits/5 · 106) of peroxidase became cell-associated by 30 min if the enzyme was presented in negatively charged liposomes (phosphatidylchloline/dicetyl phosphate/cholesterol, 70 : 20 : 10 molar ratio). Uptake of liposome-entrapped peroxidase by lymphocytes or fibroblasts was enhanced 2–5-fold if one molar percent of lysophosphatidylcholine was incorporated as a “fusogen”, and was not appreciately diminished by cytochalasin B, an inhibitor of phagocytosis. Lysophosphatidylcholine containing liposomes did not release trapped peroxidase into the medium during incubation, and studies employing the metallochromic dye, arsenazo III demonstrated lack of access of external Ca2+ to the internal, enzyme-laden, aqueous compartments; liposome-liposome fusion was also excluded by similar means. Ultrastructural cytochemstry demonstrated peroxidase within liposomes in the free cytosol of cultured cells 15–90 min after apparent liposome-cell fusion. Data provide evidence that multilamellar liposomes can be as vectors for the introjection of missing enzymes into non-phagocytic human cells.

Fibronectin-mediated cellular adhesion to vascular subendothelial matrices

Abstract Human neutrophils release lysosomal hydrolases during phagocytosis or, when phagocytosis is inhibited by cytochalasin B, upon contact with phagocytosable substances (zymosan or immune precipitates). Microtubules were more prominent in phagocytosing than in resting cells, and were observed near primary lysosomes and forming phagosomes. “Regurgitation during feeding” resulted from degranulation of primary lysosomes into newly formed phagosomes which were still open to the extracellular space as well as from the ingestion of additional material directly into already loaded secondary lysosomes. Both events led to the release of lysosomal contents from intact cells. When granule volume, calculated as the percentage of cytoplasmic volume, was determined by point counting from electron micrographs the fractional volume of granules in resting cells was 22.6% of the cytoplasm. After phagocytosing zymosan for 15 min the granule volume was reduced to 9.2%. Pretreatment with PGE 1 (2 × 10 −4 M ) or colchicine (10 −5 M ) reduced degranulation so that the fractional volume of granules in these cells after 15 min of phagocytosis was 13.4 and 11.6%, respectively. Degranulation was also inhibited by PGE 1 or colchicine when the cells ingested immune precipitates. Degranulation of lysosomes can be studied more conveniently by both morphometric and biochemical methods when cytochalasin B (5 μg/ml) transforms the leucocyte into a secretory cell in which lysosomes fuse directly with the plasma membrane as if to a phagocytic vacuole. Stimulation of cytochalasin B-treated neutrophils with zymosan led to a reduction of granule volume within the cell, and histochemically identifiable myeloperoxidase appeared at the cell/zymosan interface, indicating that lysosomes fused with the plasma membrane instead of with phagocytic vacuoles. As they did in ordinary phagocytic cells, colchicine, vinblastine, PGE 1 plus theophylline, as well as dibutyryl cAMP plus theophylline inhibited fusion of PMN granules with the plasma membrane; e.g., the mean granule volume in colchicine-treated, zymosanstimulated cells was 13% and 12.8% in cAMP treated cells as compared to untreated, zymosan-stimulated cells which contained a mean of 7.6%. These experiments provide morphologic confirmation of previous suggestions that fusion of granules with phagocytic vacuoles or the plasma membrane may be modified by cyclic nucleotides and the state of assembly of microtubules.

Crystal deposition disease: Diagnosis by electron microscopy

Abstract Although fibronectin has been well established as a mediator of cell adherence in vitro and has therefore been presumed to play a role as such in vivo, the ability of fibronectin, laid down in vivo, to mediate cell attachment, has not been demonstrated. We have therefore developed a method for quantitating the attachment of cells to vascular endothelium and subendothelial matrix and have used it to assess the role of fibronectin in promoting the attachment of a fibronectin-deficient transformed cell to this site. Whereas only 10% of cells adhered to intact endothelial linings, this attachment increased 8–10-fold when the lining cells were removed by procedures which permitted the retention of subendothelial fibronectin. When lining cells were removed by more drastic procedures which also removed subendothelial fibronectin, adherence of transformed cells was decreased to 6%. Adherence of transformed cells to the basal lamina was not, however, exclusively dependent upon fibronectin. Treatment with antifibronectin antiserum which completely inhibited adherence of these cells to the extracellular matrices of endothelial cells or fibroblasts grown in vitro could inhibit attachment to basal lamina by only 60%. The data suggest that cellular adhesion to microexudates produced by cells in culture may not be an adequate model with which to study cell adhesion in vivo. The method we describe here may provide a more realistic model and could be used to study a variety of cell-substrate interactions.

Leukocytes as secretory organs of inflammation

The diagnosis of gout and pseudogout has traditionally been established by the identification, in synovial fluid, of monosodium urate and calcium pyrophosphate dihydrate crystals with compensated polarizing light microscopy. In this paper the utility of electron microscopy in establishing these diagnosis in two cases, when the conventional means of synovial fluid analysis had failed to do so, is discussed. The application of ultrastructural analysis of synovial fluid increases diagnostic capability in the crystal deposition diseases, and it is recommended for those patients in whom the more usual studies have not established a diagnosis.

Uptake of enzyme-laden liposomes by animal cells in vitro and in vivo.

Selective release of inflammatory materials from leukocyte lysosomes may be regulated by antagonistic effects of cyclic 3′, 5′-adenosine monophosphate (cAMP) and cyclic 3′, 5′-guanosine monophosphate (cGMP). Lysosomal enzymes are released in the absence of phagocytosis when cytochalasin B (5 μg/ml) converts polymorphonuclear leukocytes (PMN) to secretory cells: lysosomes merge directly with the plasma membrane upon encounter of PMN with zymosan, and cells selectively extrude substantial proportions of lysosomal, but not cytoplasmic enzymes. β-Adrenergic stimulation (>10−8M isoproterenol or 10−7M epinephrine) of human leukocytes produced a dose-related reduction in β-glucuronidase release (blocked by 10−6M propranolol) whereas α-adrenergic stimulation (phenylephrine + propranolol) was ineffective. In contrast, the cholinergic agonist carbamylcholine chloride (>10−8M) enhanced enzyme secretion, an effect blocked by 10−6M atropine. Incubation of cells with exogenous cAMP or with agents that increase endogenous cAMP levels (dibutyryl cAMP, prostaglandin E1, histamine) reduced extrusion of lysosomal enzymes; in contrast, exogenous cGMP increased β-glucuronidase release. Whereas colchicine (5×10−4M), a drug which impairs microtubule integrity, reduced selective enzyme release, deuterium oxide, which favors microtubule assembly, enhanced selective release of lysosomal enzymes. The data suggest that granule movement and acid hydrolase release from leukocyte lysosomes requires intact microtubules and may be modulated by adrenergic and cholinergic agents which provoke changes in concentrations of cyclic nucleotides.

Chapter 17 The Role of Microtubules and Microfilaments in Lysosomal Enzyme Release from Polymorphonuclear Leukocytes

Defined, human genetic diseases due to the absence or malfunction of cytoplasmic enzymes can be divided into two main clusters. In the first, absence of appropriate hydrolytic enzyme activity ordinarily found within the vacuolar apparatus leads to the accumulation of uncleaved substrate in the lysosomes of affected tissue. Such diseases include Tay-Sachs disease, Gauchers disease, peroxidase deficiency, and various mucopolysaccharidoses. Among the second group, in which enzyme activity is missing not from the lysosomes, but from non-membrane-bound portions of the cytosol, are adenosine deaminase deficiency and the Lesch-Nyhan syndrome. Since lysosomal storage diseases are caused by a genetic deficiency of specific acid hydrolases in lysosomes, reversal of such enzyme deficiencies has been approached by means of direct enzyme replacement. Because the affected system is the lysosomal apparatus of cells, which normally takes up extracellular macromolecules and particles by endocytosis, attempts have been made to mobilize the stored GM,-ganglioside present in Tay-Sachs disease by direct administration of purified enzyme.l Unfortunately, the injected enzyme disappears rapidly from the circulation and most of it becomes localized in the liver rather than at other affected sites, such as the central nervous system. Similar results are obtained when glucocerebrosidase and ceramidetrihexosidase are 3 The problems associated with the direct administration of enzyme include: (1) the inability of this method to direct the enzyme to the parenchymal tissues containing the stored material; (2) the potential antigenicity of the enzyme; (3) the introduction of enzymes directly into the circulation where they may pre-emptively interact with their substrates or other proteins; and (4) inability of free enzyme to cross the blood-brain barrier. The genetic disorders of lysosomes and attempts to correct them, using enzyme-replacement therapy, have recently been re~iewed.~? A most promising development in the field of enzyme-replacement therapy has been the use of artificial lipid vesicles for the entrapment of enzymes and

Leucocytes as Secretory Organs of Inflammation: Control by Cyclic Nucleotides and Autonomic Agonists

Publisher Summary This chapter discusses the role of cytoskeletal elements and calcium in polymorphonuclear leukocytes (PMNs) prior and after the lysosomal enzyme release by ultrastructural means. PMNs are dramatically altered when exposed to chemotactic stimuli in the absence of cytochalasin B. Seconds after exposure to such stimuli, the PMN becomes polarized. The cytocenter—in which are found all the cells visible microtubules and the bulk of its granule population—appears to constrict, moving the granules closer to each other and to the centrioles. Morphometric methods of analysis showed that microtubules are present in greater numbers in cells stimulated to secrete than in resting cells. The chapter discusses the effects of cyclic nucleotides and colchicine and the role of calcium in lysosomal enzyme release. Electron microscopic stereology demonstrates that lysosomal enzyme release in response to receptor-ligand interactions is associated with the assembly of microtubules. Colchicine concentrations that affect microtubule assembly also inhibit lysosomal enzyme release in a dose-related fashion, but concentrations that cause the virtual disappearance of microtubules inhibit enzyme release by no more than 40%.