Heterogeneous effects of protamine on human mast cells and basophils

To investigate the mechanisms of anaphylactoid reactions to protamine, we have examined the in vitro effects of increasing concentrations of protamine (10(-6)-3 x 10(-4) mol litre-1) on the release of preformed (histamine and tryptase) and de novo synthesized (peptide leukotriene C4 (LTC4) or prostaglandin D2 (PGD2)) mediators from human basophils and mast cells isolated from lung parenchyma, heart, skin and synovial tissues. Protamine 10(-6)-3 x 10(-4) mol litre-1 induced release of histamine, but not de novo synthesis of LTC4 from basophils. At concentrations from 10(-5) to 3 x 10(-4) mol litre-1 it induced histamine release from human heart (mean 6.5 (SEM 1.5)%), skin (17.7 (4.1)%) and to a lesser extent from synovial mast cells, but not from lung mast cells. Protamine also caused the release of tryptase from heart mast cells (12.8 (3.2) micrograms/10(7) cells), but did not induce de novo synthesis of LTC4 and PGD2 from lung and skin mast cells. In these experiments cross-linking of IgE by anti-IgE caused release of LTC4 or PGD2 from human basophils or mast cells. These results demonstrate that protamine acted as an incomplete secretagogue, causing the release of preformed mediators from human basophils and mast cells.     

Apoptotic effects of different drugs on cultured retinoblastoma Y79 cells

This paper deals with the apoptotic effect exerted in human retinoblastoma Y79 cells by a number of compounds. A remarkable effect was observed after treatment with DNA-damaging agents, such as camptothecin, etoposide, cisplatin and carboplatin; camptothecin was found to be the most efficacious. Treatment with these compounds induced the appearance of morphological features of apoptosis in the cells together with the distinct fragmentation of DNA, as shown by agarose gel electrophoresis. These effects were also accompanied by a remarkable increase in the level of p53. Many other compounds, which are not DNA-damaging agents, induced the morphological features of apoptosis but none of them were capable of increasing the level of p53. Among these compounds, Taxol, suramin and sodium butyrate also stimulated the oligonucleosomal fragmentation of DNA, while C2-ceramide, a cell-permeable analogue of ceramide, and vitamin D3 were not effective in the induction of DNA laddering in Y79 cells. Apoptosis was dependent on macromolecular synthesis with all the compounds tested.     

Inhibitory effects of apple polyphenol on induced histamine release from RBL-2H3 cells and rat mast cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC50 values for histamine release were 30 micrograms/ml and 25 micrograms/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Histamine release from rat mast cells induced by the metabolic activation of drugs of abuse into free radicals

Techniques such as positron-emission tomography, single-photon-emission computed tomography, functional magnetic-resonance imaging and magnetoencephalography permit the observation of biological processes in the brain in a noninvasive manner. They have yielded new insights into the biological interrelations of sensory, motor and cognitive functions, as well as into brain diseases. Combined use of these techniques may provide more information than just the sum of its constituents, and this may narrow the gap between the biological data provided by these techniques and the mental models described by clinicians, mathematicians, psychologists and philosophers.     

Development of human mast cells from their progenitors

Two types of human mast cells, which are morphologically similar to skin mast cells and lung mast cells, respectively, can be developed from pluripotent stem cells under different culture conditions. The major growth factor for mast-cell development is c-kit ligand, which induces mastocytosis in vivo. However, this cytokine is not sufficient for full maturation of the cells.     

The ex vivo effect of the herbal medicine sho-saiko-to on histamine release from rat mast cells

OBJECTIVE: This report describes subcutaneous sarcoidosis, focusing on the radiological and magnetic resonance (MR) features of the disease. DESIGN AND PATIENTS: The cases of four patients (one male and three female, age range 36-75 years) who had subcutaneous sarcoidosis with no other organs affected were reviewed. Lesions were nodular in two cases, and in the other two were diffuse. RESULTS: Computed tomography (CT) demonstrated a well-defined, homogeneous, and enhanced lesion in the nodular cases. However, in the diffuse cases, CT showed a heterogeneous, honeycomb-like appearance and little enhancement. Angiography showed a fine stain in the arterial phase. MR imaging of the nodular lesions was homogeneous with a signal intensity similar to muscle on T1-weighted images but heterogeneous with a higher signal than muscle on T2-weighted images. Diffuse lesions showed a striped or mesh pattern with intermediate signal intensity on both T1- and T2-weighted images. Contrast-enhanced MR images showed slight enhancement. CONCLUSIONS: Subcutaneous sarcoidosis should be considered in the differential diagnosis when a patient presents with the radiological and MR features described.     

The effect of nitric oxide on cytokine-induced release of PGE2 by human cultured astroglial cells

Articles in this issue by A. C. Casiglia, A. LoCoco, and C. Zappulla; D. S. Crystal, H. Watanabe, K. Weinfurt, and C. Wu; M. Keller, N. Edelstein, C. Schmid, F. Fang, and G. Fang; and J. J. Han, M. D. Leichtman, and Q. Wang are discussed according to (a) the extent to which cultural variability can be reconciled with developmental theory and (b) the dimensions of cultural variability that matter most for development. It is argued that (a) cross-cultural research needs to be predicted on a model of how culture interacts with the forces that underlie and guide development and (b) the interpretation of cross-cultural research is severely limited without the direct measurement of the specific culture-related variables and processes that are hypothesized to account for diversity in development. Finally, within-culture variability needs to be studied in conjunction with between-culture variability so that a full model of diversity and development can be constructed.     

Inhibitory effect of alginic acids on hyaluronidase and on histamine release from mast cells

The effects of various types of alginic acid consisting of L-guluronic acids (G) and D-mannuronic acids (M) on hyaluronidase and mast cell degranulation were examined. Alginic acid with an M/G ratio of 1.0 exhibited the strongest inhibition of both activities, the higher molecular weight alginic acids of 150 to 370 kDa being preferable in both cases. Esterification of the carboxyl residue enhanced the latter activity.     

Effects of dental amalgam and its components of histamine release from human basophils and tissue mast cells

Recent studies have shown that metal ions can be released from dental amalgam or other dental materials, and can cause toxic effects on various cells. In this study, the effects of amalgam-conditioned culture medium (ACCM), components of amalgam (Ag+, Cu2+, Sn2+, Hg2+) and dental composite-conditioned culture medium (CCCM) on histamine release from human blood basophils (healthy subjects, n = 3) and tissue mast cells (n = 3) were analyzed. ACCM and CCCM were prepared using either fresh or 6-weeks-aged specimens. Of the metal ions tested, Ag+, and Hg2+ were found to induce histamine release from basophils (Ag+, 0.33 mM: 83 +/- 11% vs Hg2+, 0.33 mM: 100% vs control medium: 5 +/- 5%) and mast cells (Ag+, 0.33 mM: 91 +/- 16% vs Hg2+, 0.33 mM: 99 +/- 1% vs control: 2 +/- 1%), whereas no effects were seen with Cu2+ and Sn2+. Neither ACCM from freshly prepared amalgam nor ACCM from 6-weeks aged amalgam, produced histamine release in basophils or mast cells. Inductively coupled plasma atomic emission spectrometry (ICP) revealed that the Ag(+)- and Hg(2+)-concentrations in ACCM were below the range in which histamine release occurred. Similar to ACCM, no effects on basophils or mast cells were observed with CCCM. In summary, our data show that distinct metal ions present in dental amalgam, can induce (toxic) histamine liberation from basophils and mast cells. However, the amounts of metal ions released from amalgam apparently were too low, to cause histamine release.     

Cytokine-mediated growth hormone release from cultured ovine pituitary cells

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor alpha (TNF), recombinant ovine (ro) interleukin-1alpha (IL-1alpha), roIL-1beta, ro interleukin-2 (IL-2), and ro gamma-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1alpha and IL-1beta were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1alpha and IL-1beta stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1alpha nor IL-1beta had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1beta increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.     

Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells

KleinJan et al. (Allergy 1996;51:614-20) reported that Carnoy's fixative reduced the number of chymase-positive mast cells in the nasal mucosa. Therefore, in the present study, we investigated whether Carnoy's fixative reduces the number of chymase-positive cells from cord-blood-derived human cultured mast cells when compared with other types of fixatives. Human mast cells were obtained by culturing cord-blood-derived CD34-positive cells in the presence of stem cell factor and interleukin-6. Staining procedures of the cells in fixation with Carnoy's fixative and with other fixatives gave no differences among the number of tryptase-positive cells, whereas fixation with Carnoy's fixative for 15 min gave a significant decrease in the number of chymase-positive cells compared with acetone for 10 min. The number of chymase-positive cells decreased in a time-dependent manner under fixation with Carnoy's fixative, indicating that Carnoy's fixative had a negative effect on the number of chymase-positive cells from cord-blood-derived human cultured mast cells.     

Fucoidan, as heparin, induces tissue factor pathway inhibitor release from cultured human endothelial cells

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has antithrombotic properties, the mechanism of which is not yet completely understood. Tissue factor pathway inhibitor (TFPI), which regulates the tissue factor-dependent pathway of blood coagulation, is released from the endothelium by heparin, a mechanism contributing to its antithrombotic activity. In this study, we demonstrated that fucoidan, as heparin, induces TFPI release from cultured human umbilical vein endothelial cells (HUVEC). The TFPI accumulation in the HUVEC supernatants depends on the incubation time and polysaccharide concentration. After 30 to 60 minutes of incubation, TFPI concentration (total antigen level) was twice higher in the presence of both polysaccharides than in their absence. After one hour of incubation, in the presence of increasing concentrations of each polysaccharide, an optimal stimulation was observed for 0.5 microg/ml of fucoidan and 5 microg/ml of heparin, as evidenced by a raise of the basal TFPI level: a 2-fold increase for the total antigen and a 3-fold increase for the free antigen. These data suggest that TFPI released from vascular endothelial cells may contribute to the antithrombotic effect of fucoidan.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Effect of contignasterol on histamine release induced by anti-immunoglobulin E from rat peritoneal mast cells

In rat peritoneal mass cells induced by anti-immunoglobulin E (anti-IgE), contignasterol (1) inhibited histamine release in a dose-dependent manner. On the other hand, a reduction product of contignasterol (2) did not inhibit histamine release from mast cells induced by anti-IgE.     

Early and late events in Fc epsilon RI signal transduction in human cultured mast cells

Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cross-linking of Fc epsilonRI in rodent mast cells. To investigate the mechanism of Fc epsilonRI signal transduction in human mast cells, we used human cultured mast cells (HCMC) generated from cord blood cells in the presence of recombinant human stem cell factor and IL-6. We found that on cross-linking of Fc epsilonRI: 1) HCMC released histamine; 2) rapid tyrosine phosphorylation of multiple cellular substrates, including Syk, HS1, c-Cbl, ERK-1, and ERK-2, was observed; 3) intracellular Ca2+ and inositol phosphate production were increased within the first minute after Fc epsilonRI cross-linking; and 4) genistein, a tyrosine kinase inhibitor, inhibited both protein tyrosine phosphorylation and histamine release in a dose-dependent manner. These results were consistent with previous studies in rodent mast cells. In contrast, no tyrosine phosphorylation of phospholipase C gamma1 and Btk (Bruton's tyrosine kinase) were observed in our experimental conditions. These results suggest that the greater part of the early and late signaling events in HCMC is similar to those obtained with rodent mast cells and indicated that the requirement of tyrosine phosphorylation in the activation process of each of the signaling molecules might be different in HCMC and rodent mast cells. Our finding indicates that HCMC may be useful for analysis of Fc epsilonRI-mediated signal transduction in human mast cells.     

Manganese uptake and release by cultured human hepato-carcinoma (Hep-G2) cells

The liver is the primary organ involved in manganese (Mn) homeostasis. The human hepato-carcinoma cell line, Hep-G2, shows many liver specific functions. Consequently, Hep-G2 cells were investigated as a possible model of hepatic metabolism of Mn. Initial experiments showed that the concentration of Mn in the diet, or culture medium, similarly affected the retention of Mn by isolated rat hepatocytes and Hep-G2 cells. Manganese uptake by Hep-G2 cells suggested that uptake was followed by release from the cell. Uptake was saturable and half-maximal at 2.0 micromol Mn/L, and was inhibited by iodoacetate, vanadate, cold, and bepridil. The cations Fe2+, Cu2+, Ni2+, Cd2+, and Zn2+ decreased Mn uptake. Uptake was dependent on Calcium (Ca) concentration in a manner that resembled saturation kinetics. Cells that were pulsed with 54Mn and then placed into nonradioactive medium quickly released a large portion of their internalized Mn. Release of internalized Mn could be inhibited by low temperature, nocodozole, quinacrine and sodium azide. These data show that Hep-G2 cells are a potentially good model of hepatic Mn metabolism. Mn is taken up by a facilitated process that may be related to Ca uptake. Release apparently is an active, controlled process, that may involve microtubules and lysosomes.     

Heterogeneity of human mast cells based on cytokine content

Human mast cells can be divided into two distinct phenotypes based on their content of neutral serine proteases, suggesting that they serve differing biologic and pathologic roles. Recently, it has been demonstrated that human mast cells are a source of several pleiotropic cytokines including IL-4, IL-5, IL-6, IL-8, and TNF-alpha, but not all mast cells contain all of these cytokines, suggesting that there is also functional heterogeneity with respect to cytokine expression. In this study, we have examined the relationship between mast cell neutral protease expression and cytokine content using immunohistochemistry. Bronchial mucosal biopsies from five normal subjects and five patients with allergic asthma, and nasal mucosal biopsies from five normal subjects and three patients with allergic rhinitis were embedded in glycol methacrylate. Sections (2 microns) were stained for IL-4, IL-5, and IL-6, adjacent to serial sections stained for tryptase and chymase. The distribution of cytokines among the tryptase+ chymase- mast cells (MCT) and tryptase+ chymase+ mast cells (MCTC) was examined by co-localization of cytokines to MCTC or MCT in serial sections using the camera-lucida. Although IL-4 was distributed among both mast cell phenotypes, it was expressed preferentially by the MCTC subset (overall 85% MCTC:15% MCT). In contrast, IL-5 and IL-6 were restricted almost exclusively to the MCT subset. Immunostaining of isolated skin mast cells (> 99% MCTC) supported these findings, with strong immunoreactivity present for IL-4 but very little for IL-5 or IL-6. These results indicate that in addition to exhibiting heterogeneity with respect to neutral protease content of the secretory granules, human mast cells are also heterogeneous with respect to cytokine content. This suggests that the biologic functions of MCTC and MCT cells differ as a result of their capacity to generate and release different cytokine profiles.