Sympathetic nerve contact causes maturation of mast cells in vitro

Using a tissue culture model developed to study interactions between peripheral neurons and mast cells (MC), time-lapse microscopy showed that RBL- 2H3 cells (a model of the mucosal MC) formed attachments with sympathetic neurons, ceased to divide, and moved along neurites toward the cell bodies. Electron microscopy showed significant increase in granules compared to intrinsic controls (RBL cells in coculture but lacking neurite contact). In studies using cohort cultures of 12- to 14-day-old sympathetic neurons, RBL cells adhered more rapidly to neurons than did control YB2/0 cells (a neutral target cell), and were inhibited in growth compared with RBL cells cultured in parallel without neurons. RBL cells cocultured with neurons for 24–48 h took up significantly more ³H-5HT and released a significantly larger percentage of ³H-5HT in response to the calcium ionophore A23187 than RBL cells in parallel pure cultures. Since no change in MC phenotype was seen, we conclude that contact with nerve membrane may be a developmental cue leading to maturation of MC. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 173–182, 1998     

Localization of SSeCKS in unmyelinated primary sensory neurons

Background

RhoA and Rho kinase inhibitors overcome the inhibition of axonal regeneration posed by central nervous system (CNS) substrates.

Methods

To investigate if inhibition of the Rho pathway augments the neurite extension that naturally occurs in the peripheral nervous system (PNS) following nerve damage, dorsal root ganglion neurons and Schwann cell co-cultures were incubated with culture medium, C3 fusion toxin, and the Rho kinase (ROCK) inhibitors Y27632 and H1152. The longest neurite per neuron were measured and compared. Incubation with Y27632 and H1152 resulted in significantly longer neurites than controls when the neurons were in contact with Schwann cells. When separated by a porous P.E.T. membrane, only the group incubated with H1152 developed significantly longer neurites. This work demonstrates that Rho kinase inhibition augments neurite elongation in the presence of contact with a PNS-like substrate.     

A chymotrypsin-type serine protease in rat basophilic leukemia cells: evidence for its immunologic identity with atypical mast cell protease

Activity of a chymotrypsin-type serine protease was found in a subline of rat basophilic leukemia (RBL-2H3) cells. The protease was immunologically cross-reactive with anti-atypical mast cell protease immunoglobulin (Ig) G, and its activity was inhibited in a dose-dependent manner by the antibody. The apparent m.w. of the protease that reacted with the antibody was 25,000, which was identical with that of atypical mast cell protease in rat mucosal mast cells. These results show that the chymotrypsin type serine protease in RBL-2H3 cells is immunologically identical with atypical mast cell protease, which was first purified from rat small intestine. Immunohistochemical studies showed that the protease was located not only in intracytoplasmic granules but also in organelles synthesizing protein, such as cisternae of the rough endoplasmic reticulum, perinuclear spaces, and the Golgi apparatus. However, no immunoreactivity was demonstrated in rat basophils. The activity of the protease increased in the exponential phase of growth of RBL-2H3 cells in which some activity was also detected in the medium, and it decreased in the late stationary phase.     

Identification of anti-allergic effect of Clonorchis sinensis-derived protein venom allergen-like proteins (CsVAL)

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases.     

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.     

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 μg/ml and 25 μg/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.     

Neuronal Survival,Morphology and Outgrowth of Spiral Ganglion Neurons Using a Defined Growth Factor Combination

Objectives

The functionality of cochlear implants (CI) depends, among others, on the number and excitability of surviving spiral ganglion neurons (SGN). The spatial separation between the SGN, located in the bony axis of the inner ear, and the CI, which is inserted in the scala tympani, results in suboptimal performance of CI patients and may be decreased by attracting the SGN neurites towards the electrode contacts. Neurotrophic factors (NTFs) can support neuronal survival and neurite outgrowth.

Methods

Since brain-derived neurotrophic factor (BDNF) is well known for its neuroprotective effect and ciliary neurotrophic factor (CNTF) increases neurite outgrowth, we evaluated if the combination of BDNF and CNTF leads to an enhanced neuronal survival with extended neurite outgrowth. Both NTFs were added in effective high concentrations (BDNF 50ng/ml, CNTF 100ng/ml), alone and in combination, to cultured dissociated SGN of neonatal rats for 48 hours.

Results

The neuronal survival and neurite outgrowth were significantly higher in SGN treated with the combination of the two NTFs compared to treatment with each factor alone. Additionally, with respect to the morphology, the combination of BDNF and CNTF leads to a significantly higher number of bipolar neurons and a decreased number of neurons without neurites in culture.

Conclusion

The combination of BDNF and CNTF shows a great potential to increase the neuronal survival and the number of bipolar neurons in vitro and to regenerate retracted nerve fibers.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca²⁺ level ([Ca²⁺]_i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca²⁺]_i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca²⁺]_i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca²⁺]_i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca²⁺]_i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca²⁺ signaling and degranulation. mast cell; immunoglobulin E; cytochalasin D; Y-27632; wortmannin     

Rodent CNS neuroblasts exhibit both perpendicular and parallel contact guidance on the aligned parallel neurite bundle.

Mouse cerebellar granule cells showed two types of migration behavior in microexplant cultures. They first migrated along their neurites, showing the typical contact guidance, and then oriented themselves at right angles to the parallel neurites, thus exhibiting the 'perpendicular contact guidance' (Nakatsuji, N. and Nagata, I. 1989 Development, 106, 441-447). To study whether other neurons have the capacity to show similar 'perpendicular contact guidance', we cultured dissociated neuroblasts from various parts of CNS or PNS on parallel neurite bundles. The PNS neuroblasts always extended their processes parallel to the neurite bundle. In contrast, almost all kinds of CNS neuroblasts tested oriented their processes both perpendicular and parallel to the neurite bundles that were all free of glia. Time-lapse video recording revealed that neuroblasts migrated in both directions. Thus, CNS neuroblasts possess the capacity to migrate and extend their processes at right angles to the substratum of heterotypic neurite bundles, which may play an important role in histogenesis of the CNS during development.     

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcRI, LAT and α-galactosyl derivatives of ganglioside GD_1b mobilized to lipid raft domains following FcRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of β-hexosaminidase activity after FcRI activation. The two mutant cell lines have a reduced release of β-hexosaminidase activity after FcRI stimulation, but not after exposure to calcium ionophore. These results indicate that the α-galactosyl derivatives of ganglioside GD_1b are important in the initial events of FcRI signaling upstream of Ca²⁺ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcRI.     

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite

Mast cell-neurite interaction serves as amodel for neuroimmune interaction. We have shown that neurite-mast cellcommunication can occur via substance P interacting with neurokinin(NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilicleukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both ofwhich activate only neurites. Cell activation was assessed by confocalimaging of Ca²⁺ (cells preloaded with fluo 3), and analysesof RBL CD63-GFP⁺ granule movement were conducted. Neuriteactivation by BK or SV was followed by RBL Ca²⁺mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not onnoncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 ± 0.04 vs. 0.05 ± 0.01 µm/s) thatwas most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of suchprecise (restricted to the membrane's contact site) transfer ofinformation between nerves and mast cells that could allow for verysubtle in vivo communication between these two cell types.

     

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.     

Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.     

Agonists of the MAS-related gene (Mrgs) orphan receptors as novel mediators of mast cell-sensory nerve interactions

IgE-dependent activation of mast cell activation is often associated with symptoms attributed to activation of sensory nerves. Depending on the tissues involved such symptoms include itching, sneezing, irritation, vasodilation, and reflex secretions. In the present study, we hypothesize that sensory neuroactive mediators released from mast cells may include agonists of recently discovered orphan receptors referred to as sensory nerve specific receptors or products of mas related genes. HEK-293 cells expressing MrgC11 receptors and wild-type HEK-293 cells were loaded with the calcium indicator Fura-2. A known stimulant of MrgC11 receptors the RF-amide, neuropeptide FF, evoked a rapid increase in cytosolic calcium in the MrgC11 expressing cells but not in the wild-type HEK-293 cells. IgE-dependent stimulation of either rat basophilic leukemia-2H3 cells (RBL-2H3 cells) or mouse bone marrow-derived mast cells, released a substance(s) that stimulated increases in cytosolic calcium in the MrgC11 expressing cells that far exceeded that seen in control cells. RT-PCR revealed that both mouse mast cells and RBL-2H3 cells express the RF-amide precursor gene proneuropeptide FF (A). Immunohistochemical analysis demonstrated RF-amide immunoreactivity in mouse skin mast cells in situ and in mast cells isolated from mouse skin. These data support the hypothesis that agonists of certain sensory nerve specific receptors or mas related genes may participate in mast cell sensory nerve interactions.     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Application of on-chip cell cultures for the detection of allergic response

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.