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PubMed1987

The T cell-specific serine proteinase TSP-1 is associated with cytoplasmic granules of cytolytic T lymphocytes.

This study describes the localization of the previously purified T cell-specific serine proteinase, termed TSP-1 (M. M. Simon et al., EMBO J. 1986. 5: 3267), within cytoplasmic granules of cytolytic T cell lines (CTLL). Subcellular fractionation of disintegrated CTLL (ruptured by nitrogen cavitation) was accomplished by Percoll density gradient centrifugation of cell lysates (postnuclear supernatant). Individual fractions were tested for proteinase activity on chromogenic peptide substrates and for the presence of TSP-1 by Western blot analysis. In addition, each fraction was assayed for cytolytic activity against sheep red blood cells (SRBC), for protein and for additional marker enzymes to assess the enrichment for cellular organells. All serine enzyme-type molecules including TSP-1 expressed by CTLL were identified by labeling cell lysates or gradient fractions with the serine proteinase-specific affinity ligand tritiated diisopropyl fluorophosphate [( 3H]DFP) in the presence or in the absence of class-specific or enzyme-specific proteinase inhibitors and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data demonstrate that the Percoll gradient fraction, which was shown by morphological examination in the electron microscope to be highly enriched for cytoplasmic granules, also contained greater than 80% of proteinase activity in addition to the granule-associated structures cytolysin and arylsulfatase. The identity of the granule-associated proteinase in two independent cell lines, CTLL HY3-Ag3 and CTLL 1.D.9, with the serine proteinase TSP-1 is indicated by its specificity for the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, its sensitivity to class-specific as well as TSP-1-specific enzyme inhibitors and by its reactivity with a polyvalent TSP-1-specific rabbit antiserum. Both CTLL contain a [3H]DFP-labeled protein that migrates with a molecular mass of 60 kDa under nonreducing conditions and with 30 kDa under reducing conditions and which can be inactivated by the TSP-1-specific inhibitor H-D-Pro-Phe-Arg-chloromethylketone. CTLL HY3-Ag3 (a long-term culture CTLL with natural killer-like activity) but not CTLL 1.D.9 (an antigen-specific short-term cultured CTLL) express in addition a further [3H]DFP-binding protein which migrates with 27 kDa under nonreducing or reducing conditions. No substrate specificity was found for this molecule. The possible function of the granule-associated serine proteinase TSP-1 is discussed.

Amides↗
PubMed2001

Staining pattern of the brush border and detection of cytoplasmic granules in the uriniferous tubules of female DBA/2Cr mouse kidney: comparison among various fixations and stains.

The proximal straight tubules of the female mouse kidney exhibit heavy periodic acid Schiff (PAS) staining in their brush borders and numerous cytoplasmic granules. In the present study, the female DBA/2Cr mouse kidney was examined, using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin, or Carnoy solution) and various staining methods (HE, PAS, alcian blue, periodic acid methenamine-silver (PAM), toluidine blue, azan, or Congo red). Under azan and PAM, the staining pattern of the brush border was similar to that of PAS, and few effects of the fixative were observed. Cytoplasmic granules were clearly detected with PAM as well as PAS. However, these granules were not detected with Carnoy solution. Furthermore, distribution of granules differed between PAS and PAM.

Animals↗
PubMed2002

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity.

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.

Animals↗
PubMed2004

The beta-N-acetylhexosaminidase of Entamoeba histolytica is composed of two homologous chains and has been localized to cytoplasmic granules.

We have purified a beta-N-acetylhexosaminidase from trophozoites of Entamoeba histolytica to homogeneity. In SDS-PAGE, the enzyme yielded a single protein band at an apparent M(r) of 64,000. The elution behaviour of the native enzyme upon molecular sieve chromatography corresponded to a molecular mass of approximately 132,000 suggesting that the enzyme is a dimer. Upon sedimentation velocity centrifugation, hexosaminidase activity sedimented at 12S, implying aggregation to a higher molecular mass complex with an apparent M(r) of approximately 400,000. Based on the N-terminal sequence of the purified enzyme and on data extracted from the E. histolytica genomic data base, we amplified and cloned two genes (EhHEXA and EhHEXB) coding for two presumptive, highly similar hexosaminidase chains which we designated as Ehhexalpha and Ehhexbeta. Northern blot analysis indicated that the two genes were expressed to a similar level, and Western blotting with chain-specific antisera showed that the trophozoites synthesize both proteins. By cell fractionation, the hexosaminidase was found to be a major component of cytoplasmic granules; these contain tissue-destructive factors and are released after collagen-induced exocytosis to the cell surface. In agreement with this observation, immunocytochemistry with an antiserum cross-reacting with both hexosaminidase chains revealed strong fluorescence in surface patches, which we interpret as released granules, and in vesicles throughout the cell. Its localization in cytoplasmic granules strengthens the notion that the hexosaminidase complex may contribute to amoebic pathogenicity.

Amino Acid Sequence↗
PubMed1989

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells.

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.

Animals↗
PubMed2002

Modification of actin, myosin and tubulin distribution during cytoplasmic granule movements associated with platelet adhesion.

BACKGROUND AND OBJECTIVES: Cytoskeletal elements determine the changes in platelet cell shape which occur during adhesion, aggregation and release of granular contents as part of the activation process. The aim of this study was to characterize the changes in the distribution of actin filaments, myosin and tubulin molecules during several stages of platelet adhesion to glass and their association with granule displacement, as assessed by confocal microscopy. DESIGN AND METHODS: Platelets obtained from healthy donors were adhered to glass and cytoskeleton distribution was characterized and correlated to changes of cell shape and intracellular granule displacement by immunofluorescence assays and phase contrast microscopy. Treatment with specific cytoskeleton inhibitors such as cytochalasin D, butanedione monoxime and colchicine were used before and after the adhesion process. The spatial distribution of the cytoskeleton in association with cytoplasmic granules was analyzed in both confocal microscopy projections and three-dimensional images obtained by merging the respective projections. RESULTS: Our experiments revealed that as platelets contact the substrate, a sequential and simultaneous rearrangement of actin filaments, myosin and tubulin molecules occurred and this was related to cell shape, as well as to movements of cytoplasmic granules. Treatment of platelets with cytoskeleton inhibitors, modified not only the target molecule but also other cytoskeletal components with consequent alterations in the studied platelet functions. INTERPRETATION AND CONCLUSIONS: During platelet adhesion to glass and granule displacement, a close spatial and functional relation between actin filaments, myosin molecules and microtubules was observed suggesting that these different cytoskeleton components interact in supporting the platelet functions here studied.

Actins↗
PubMed1988

Self-protection of cytotoxic lymphocytes: a soluble form of homologous restriction factor in cytoplasmic granules.

A soluble form of homologous restriction factor (HRF) has been isolated from the cytoplasmic granules of human large granular lymphocytes that were cultured in the presence of recombinant interleukin 2 for 2-3 weeks. The granule-derived protein (approximately 65 kDa) is soluble in detergent-free solution and reacts with antibody produced to membrane HRF. HRF was first described as a 65-kDa membrane protein of human erythrocytes capable of inhibiting the formation of transmembrane channels by the membrane attack complex of complement. It has also been isolated from activated human lymphocytes and shown to confer upon these cells relative resistance to lysis by the membrane attack complex and by the complement component C9-related protein of human cytotoxic lymphocytes. The soluble HRF of lymphocyte granules inhibits reactive lysis of erythrocytes by the membrane attack complex of human complement. It was also found to be a potent inhibitor of (i) the cytolytic activity of the C9-related protein of human cytotoxic lymphocytes, (ii) human large granular lymphocyte cytotoxicity, and (iii) the cytotoxic activity of human CD8+ lymphocytes obtained by cell sorting from recombinant interleukin 2-activated peripheral blood mononuclear cells. It is proposed that granule-derived soluble HRF and cell surface-membrane-bound HRF are involved in the mechanism of self-protection of killer lymphocytes.

Blood Proteins↗
PubMed1982

Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.

By using fluorescent antibody staining, we have followed cytoplasmic granules unique to germ-line cells throughout the life cycle of Caenorhabditis elegans. These elements, designated P granules, are segregated exclusively to germ-line precursor cells during early embryogenesis. Prior to mitosis at each of the early cleavages that produce a somatic and germ-line daughter cell, the granules become localized in the region of cytoplasm destined for the germ-line daughter. After the 16-cell stage, the granules appear to be associated with the nuclear envelope. P granules persist in the germ cells throughout the larval and adult stages. The P granules are similar in number, size, and distribution to germ-line-specific structures identified as "germinal plasm" by electron microscopy in C. elegans embryos.

Animals↗
PubMed1988

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes.

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.

Animals↗
PubMed1994

Cytoplasmic granule formation in mouse pancreatic acinar cells. Evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size, and for reductions in membrane surface area and immature granule volume during granule maturation.

We used a computer-assisted morphometry approach to analyze quantitatively the process of cytoplasmic granule formation in mouse pancreatic acinar cells stimulated with pilocarpine to induce secretion. Our findings suggest that each condensing vacuole/immature granule of pancreatic acinar cells is formed by the progressive aggregation of 106 to 128 "unit progranules" of narrowly fixed volume, define a range of 7.7 to 9.2 for the factor of volume condensation between the largest immature granules and the mature unit granule, and predict that the formation of a single mature unit granule by the aggregation and fusion of unit progranules involves a net reduction of at least 95% in the amount of membrane surface area associated with these structures.

Animals↗
PubMed1960

The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes.

A method has been described for isolation of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. Homogeneous suspensions of leucocytes were disrupted by lysis in 0.34 M sucrose. This procedure liberated the cytoplasmic contents of the cell and dissolved a considerable proportion of the nuclei. Following disruption, the sucrose lysate was separated into three fractions by differential centrifugation, i.e. 400 g or nuclear pellet, 8,200 g or granule pellet and the postgranule supernate. Microscopic examination revealed that the 8,200 g pellet was composed of intact granules as well as occasional mitochondria. The other two fractions were morphologically heterogeneous. Studies with isolated granules demonstrated their lysis by a variety of weak acids and surface-active agents. When buffered solutions were employed between the ranges of pH 2.0 and 9.0, granule lysis began at pH 5.5 and was complete at pH 4.0. Chemical analysis disclosed that the granule pellet contained protein and phospholipid with only traces of nucleic acids. Approximately 70 to 80 per cent of the total cellular antimicrobial agent phagocytin was present in the granule fraction. This material was liberated from the granules by acid (pH 5.0 or lower). Studies on selected enzymes showed that acid phosphatase, alkaline phosphatase, nucleotidase, ribonuclease, deoxyribonuclease, and beta glucuronidase were predominantly localized in the granule fraction. Approximately 50 per cent of total cellular lysozyme and cathepsin were also present in the 8,200 g pellet. Disruption of the granules was associated with the release of the majority of granule protein and enzymes in a non-sedimentable form. The properties and composition of rabbit polymorphonuclear leucocyte granules seem to be analogous to those of liver lysosomes.

Acid Phosphatase↗
PubMed1987

A serine protease (CCP1) is sequestered in the cytoplasmic granules of cytotoxic T lymphocytes.

Based upon the use of a new predictive algorithm, three peptides were synthesized that correspond to likely antigenic sites of the cytotoxic T cell-specific protease cytotoxic cell protein 1 (CCP1). Antibodies raised against these peptides, under reducing conditions, bound to a single protein of m.w. 29,000 found in two actively cytotoxic T cells. This protein was absent from a "cytotoxic" T cell line that had lost its cytolytic properties. By using immunocytologic techniques, the protein was localized within cytoplasmic granules. In addition, granules purified from the cytoplasm of cytotoxic T cells were shown, by Western blot analysis, to contain the protein. The molecule detected by the antibodies behaves, upon reduction, similarly to a diisopropylfluorophosphate-binding protein. Thus, here we provide evidence that CCP1, a protein whose expression correlates with cytotoxicity, is contained within organelles which have been implicated in killing. These findings strongly suggest that CCP1 plays a key role in T cell-mediated target cell lysis.

Cell Line↗
PubMed1995

Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells.

The sulfhydryl agent, cysteamine (CSH), promotes the accumulation of autofluorescent, peroxidase-positive cytoplasmic granules in cultured astroglia akin to those which naturally accumulate in astrocytes of the aging periventricular brain. Both in vitro and in situ, CSH rapidly induces various heat shock proteins (HSP) in astrocytes long before granulation occurs. In the present study, we determined that CSH treatment resulted in an increase in HSP 27, HSP 90 and heme oxygenase (HO-1) at both the protein and mRNA level. We also showed that C6 glioma cells, unlike primary astrocytes, constitutively express HSP 27, HSP 90 and HO-1 at low levels. Moreover, CSH is incapable of eliciting further HSP expression or inducing granulation in the glioma cells. Our results support the hypothesis that the biogenesis of redox-active astrocytic inclusions in CSH-treated glial cultures and in the aging periventricular brain is dependent on an antecedent cellular stress response.

Animals↗
PubMed1989

Biological and clinical features of acute lymphoblastic leukaemia with cytoplasmic granules or inclusions: description of eight cases.

We describe eight patients (four children and four adults) with an acute lymphoblastic leukaemia (ALL) with cytoplasmic granules or inclusions. The incidence of this variant of acute leukaemia in our whole series of patients with ALL is 1.8%. The granules or inclusions were usually positive for aspecific esterases (ANAE) and/or acid phosphatase, and the immunophenotype was in all cases typical of a CALLA positive B-lineage ALL (CD10+, CD19+ and/or CD24+, DR+, TdT+, anti-T-, anti-My-, SIg-). In one paediatric case, CD33 was unusually coexpressed. Ultrastructural investigations were performed in one case and demonstrated large granules containing vesicles, usually membrane bound, in the majority of blast cells. In the two cases analysed, Ig heavy chain gene rearrangement was detected. In this series of patients prognosis was poor since three never achieved a complete remission, four relapsed and only one is still in first continuous remission.

Acid Phosphatase↗
PubMed1988

T helper cell cytoplasmic granules. Exocytosis in response to activation via the T cell receptor.

A series of class II MHC-restricted keyhole limpet hemocyanin-specific Th cell clones were examined for cytoplasmic granules by histochemical techniques and fractionation of their homogenates. All showed granules containing lysosomal enzymes and high levels of trypsin-like activity revealed by a N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase assay. Using the latter as a marker for granule contents, granule secretion was observed in response to MHC-restricted, Ag-dependent signals presented in vitro, and correlated well with T cell activation as measured by proliferation. However, human rIL-2, a strong inducer of T cell proliferation, did not stimulate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase secretion by itself, nor did it influence the secretion mediated by Ag. Other factors found to induce secretion included immobilized antibodies directed against determinants of the TCR complex, as well as Con A. These results suggest that granule enzyme secretion is another indicator of TCR-mediated activation and provides a possible mechanism for Th cell function via a rapid, local delivery of granule contents to adjacent cells.

Animals↗
PubMed1989

Expression of cytoplasmic granules with T cell-associated serine proteinase-1 activity in Ly-2+(CD8+) T lymphocytes responding to lymphocytic choriomeningitis virus in vivo.

Monoclonal antibodies (mAb) with specificity for the T cell-associated serine proteinase-1 of the mouse (MTSP-1) were used to study expression and storage of this enzyme in T lymphocytes during lymphocytic choriomeningitis (LCM) virus infection in vivo. Immunohistochemical analysis of splenic tissue at the peak of LCM virus-specific T cell-mediated cytolytic responses, i.e., at day 7 post infection, revealed high numbers of MTSP-1+ T lymphocytes in the interfollicular T cell-dependent area of the spleen. More than 50% of Ly-2+(CD8+) cells but only low numbers of Ly-2-(CD8-) cells, previously enriched by flow cytofluorometry, contained large amounts of cytoplasmic granules which stained both with MTSP-1-specific mAb and the esterase substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. These data demonstrate that in vivo generated LCM virus-induced cytolytic T lymphocytes develop cytoplasmic storage granules containing MTSP-1 and suggest that the mechanism of granule exocytosis is operative in vivo; possibly MTSP-1 is one effector molecule participating in the Ly-2+(CD8+) T lymphocyte-mediated control of virus infection.

Animals↗