Interactions between rat submucosal neurons and mast cells are modified by cytokines and neurotransmitters

European Journal of Pharmacology 864 (2019) 172713

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Interactions between rat submucosal neurons and mast cells are modified by cytokines and neurotransmitters

T

Jasmin Ballout, Martin Diener∗ Institute for Veterinary Physiology and Biochemistry, Justus Liebig University Giessen, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Cl-ecretion Cytokine Enteric nervous system Mast cell Nicotine

The role of mast cells during inflammatory bowel diseases (IBD) is discussed controversially. Whereas several studies report an increase in mast cell density during IBD, others found a decrease. Recently, we observed a reduced response to mast cell degranulation induced by antigen contact in a colitis model. As the effects of mast cell mediators on epithelial ion transport are mediated indirectly via stimulation of secretomotor neurons, we investigated in vitro whether proinflammatory cytokines change the response to mast cell degranulation. Tumor necrosis factor α (TNFα) and a mix of proinflammatory cytokines caused an increase of short-circuit current (Isc) and tissue conductance in rat colon. Anion secretion induced by histamine was downregulated in the presence of interleukin-1β (IL-1β) and the cytokine mix, whereas the response to the mast cell stimulator compound 48/80 was not changed significantly. In a coculture of rat submucosal ganglionic cells with a mast cell line (RBL-2H3), TNFα preincubation for 1 d increased the percentage of neurons responding to mast cell degranulation with an increase of the cytosolic Ca2+ concentration and enhanced the amplitude of this response. Consequently, the downregulation of epithelial secretion is compensated by an increased sensitivity of secretomotor neurons leading to a constant response of the epithelium to compound 48/80. Furthermore, enteric neurons can modify mast cell functions as nicotine inhibited the increase in cytosolic Ca2+ concentration of RBL2H3 cells and the Isc evoked by compound 48/80. Consequently, these in vitro models deliver new insights into cellular interactions in the gut wall under inflammatory conditions.

1. Introduction The interaction between intestinal mast cells and enteric neurons is important for different physiological functions, e.g. the induction of intestinal secretion. Even more important, this interaction plays an essential role during intestinal disorders like food allergies or inflammatory bowel diseases (IBD), where a higher density of mast cells has been observed in the intestinal wall (Bischoff, 2009). Once activated, mast cells release mediators, which are either stored in granules such as histamine, proteases, tumor necrosis factor α (TNFα) or serotonin, or are synthesized de novo such as prostaglandins, leukotrienes or several cytokines (De Winter et al., 2012). A part of the action of these mediators is mediated by stimulation of submucosal secretomotor neurons expressing receptors for histamine (histamine H1, H2 and H4 receptor) or proteases (protease-activated receptor type 1 and 2) (Buhner and Schemann, 2012; Bell et al., 2015). The release of prosecretory transmitters by these neurons close to the intestinal epithelium induces a secretion of ions and water into the gut lumen. It is thought that the higher mast cell density in the gut wall observed during IBD

∗

might maintain a vicious cycle of inflammation (Rijnierse et al., 2007). Interestingly, in a previous study we observed that in rats sensitized against ovalbumin the additional induction of a colitis by colonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) did not increase anion secretion induced by mast cell degranulation in vitro. In contrast, the secretory responses tended to be reduced both in inflamed colonic regions as well as in small intestinal segments not affected by the locally induced colitis (Becker et al., 2019). Intestinal mast cells can be stimulated, among others, by crosslinking of immunoglobulin E (IgE) antibodies, which are bound on the plasma membrane of the mast cells via the Fcε receptor (Kurashima and Kiyono, 2014). Not only this classical activation, but also IgE-independent stimuli, e. g. cytokines, neuropeptides or toxins, lead to a degranulation of mast cells (Da Silva et al., 2014). New important players in the IgE-independent mast cell activation are G protein-coupled Mas-related gene receptors, which are found on the surface of mast cells (and neurons) in a species- and tissue-specific manner. These receptors might play an important role in the mast cell-neuron interactions, especially under inflammatory conditions in the intestine, where

Corresponding author. Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Gießen, Frankfurter Str. 100, Gießen, 35392, Germany. E-mail address: [email protected] (M. Diener).

https://doi.org/10.1016/j.ejphar.2019.172713 Received 23 August 2019; Received in revised form 20 September 2019; Accepted 1 October 2019 Available online 03 October 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.

European Journal of Pharmacology 864 (2019) 172713

J. Ballout and M. Diener

2.2. Solutions

the expression of different subtypes of Mas-related gene receptors is increased (Avula et al., 2013). Mas-related gene receptors probably also mediate mast cell activation induced by the neuropeptide substance P (McNeil et al., 2015), a response which has been intensively studied as model for neuro-immune interactions (Koon and Pothoulakis, 2006; Van Nassauw et al., 2007). However, mast cells also express receptors for different neurotransmitters. For example, the catecholamine norepinephrine, which is known to modify secretory processes in distal colon of rats (Schultheiss and Diener, 2000), can activate human lung mast cells via β2-adrenergic receptors (Butchers et al., 1991). A histamine release from mast cells can be evoked by cholinergic agonists via muscarinic receptors in a dose-dependent manner (Blandina et al., 1980). Moreover, nicotinic acetylcholine receptors (α7, α9 and α10) are found on the surface of mast cells with an inhibitory effect on mast cell degranulation (Mishra et al., 2010; Yamamoto et al., 2014). Interestingly, cigarette smoking exerts a protective action against the development and the progression of ulcerative colitis, but not of Crohn's disease (Berkowitz et al., 2018). Due to these mutual interactions between mast cells and enteric neurons, which might contribute to the pathophysiology of inflammatory bowel diseases, in the present study a coculture model of rat submucosal ganglionic cells and RBL-2H3 cells (rat basophilic leukemia cells; an equivalent rat mast cell line) was used to find out whether the communication between these cell types is modified by proinflammatory cytokines or neurotransmitters. As readout, the increase in the cytosolic Ca2+ concentration in both cell types induced by the mast cell stimulating agent compound 48/80 was measured after preincubation with different proinflammatory cytokines or neurotransmitters. RBL-2H3 cells, which show functional similarities with rodent mucosal mast cells (or human tryptase-containing mast cells), have a basal sensitivity to compound 48/80 (Fowler et al., 2003; Bell et al., 2015), which is assumed to act via Mas-related gene receptors (Ali, 2017). Although it is known that the sensitivity of RBL-2H3 cells to basic secretagogues can be significantly enhanced by coculture with fibroblasts (Swieter et al., 1993) or protein kinase inhibitors like quercetin (Senyshyn et al., 1998), no attempt was made in the present study to upregulate the sensitivity as obviously under the culture conditions used the large majority of the cells could be stimulated with compound 48/80 (cf. Fig. 9). These experiments were accomplished by Ussing chamber experiments, in which the effect of degranulation of intestinal mast cells on ion secretion and permeability of mucosa-submucosa preparations from rat distal colon was tested in the absence or presence of proinflammatory cytokines and/or neurotransmitters assumed to modify mast cell degranulation. The mucosa-submucosa preparations contain spontaneously active, secretomotor neurons in the submucosal plexus (Andres et al., 1985), which are strongly activated by typical mast cell mediators such as histamine (Bell et al., 2015) or mast cell mediator cocktails released by antigen contact after prior sensitization of the animals (Javed et al., 1992; Hug et al., 1996).

For the Ussing chamber experiments, a Parsons bathing solution was used containing (in mmol/l): 107 NaCl, 4.5 KCl, 25 NaHCO3, 1.8 Na2HPO4, 0.2 NaH2PO4, 1.25 CaCl2, 1 MgSO4 and 12.2 glucose. The solution was gassed with carbogen (5% CO2 and 95% O2, v/v) and kept at a temperature of 37 °C. The standard Tyrode solution, which was used for Ca2+ imaging experiments, consisted of (in mmol/l): 140 NaCl, 5.4 KCl, 10 HEPES (N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid), 12.2 glucose, 1.25 CaCl2, and 1 MgCl2. For fixation and other solutions (blocking, antibody, washing) used for immunocytochemistry, a phosphate buffer saline (PBS; 10 mmol/l) was used. With the exception of the latter, the pH of all solutions was adjusted to 7.4 with NaOH (1 mmol/l) or HCl (1 mmol/l). For the cell culture experiments two different media were used. The isolated submucosal ganglionic cells were incubated in Neurobasal-A Medium (NB-A; Thermo Fisher Scientific) containing 10% (v/v) fetal calf serum, 1% (v/v) penicillin/streptomycin (10,000 U/ml penicillin G with 10 mg/ml streptomycine sulphate) and 2 mmol/l L-glutamine. For the mast cell line RBL-2H3, Minimum Essential Medium alpha (MEM α; Thermo Fisher Scientific) was used containing 1% (v/v) penicillin/ streptomycin, 2 mmol/l L-glutamine, and 15% (v/v) fetal calf serum. 2.3. Tissue preparation The distal colon was removed from the abdominal cave and rinsed with Parsons buffer. Muscle-stripped mucosa-submucosa preparations were used to reduce the diffusion barrier. Therefore, the distal colon was put over a plastic rod, a slight circular incision was made near the distal end of the colon and the serosa and the lamina muscularis were removed carefully by hand in proximal direction. The tissue was opened lengthwise at the mesenteric side and was cut into two segments for Ussing chamber experiments. In general, one segment from each animal served as control tissue, whereas the other was pretreated with the respective agonist/cytokine. For the isolation of submucosal ganglionic cells, the mucosa-submucosa preparation was fixed on a glass plate at their proximal end with the mucosal side at the top. After making a slight incision in the mucosa with a specimen holder, the mucosal layer was removed with the sharp side of a glass slide in proximal direction. The submucosa was cut into 2 × 2 mm squares and transferred into a 2 ml tube with cold Parsons buffer. For the experiments with intact submucosa, the submucosa was cut into 5 × 5 mm squares, which were mounted on poly-Llysine (0.1 mg/l) coated coverslips and transferred into 4-well plates. After a few minutes, the intact submucosa was washed with Tyrode solution, covered with 500 μl sterile NB-A per well and stored overnight in the incubator (37 °C, 5% (v/v) CO2). 2.4. Ussing chamber experiments The mucosa-submucosa preparations were fixed in a modified Ussing chamber, which was filled with 3.5 ml bathing solution on each side and tempered at 37 °C. For electrophysiological measurements the tissue was short-circuited by a computer-controlled voltage-clamp (Mussler, Aachen, Germany). The tissue conductance (Gt; in mS/cm2) was measured every minute. The short-circuit current (Isc) in μEq/h/ cm2 (with 1 μEq/h/cm2 = 26.9 μA/cm2) expresses the flux of monovalent ions per time and area. Secretion of anions (or electrogenic absorption of cations) is reflected by a positive Isc. Experiments were started after a stabilization phase of about 60 min. In parallel to a time-dependent control tissue, one tissue from each rat was preincubated for 1 h with a proinflammatory cytokine, i.e. tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β) or interferon γ (IFN- γ), or a mix of these cytokines for up to 3 h. Afterwards, all tissues were stimulated with the mast cell activator compound 48/80 and histamine separated by a washing step (3 × washing of the serosal

2. Material and methods 2.1. Animals Male and female Wistar rats were used for the experiments ranging from 8 to 12 weeks for the Ussing chamber and Ca2+ imaging experiments and 5–7 weeks for the isolation of submucosal ganglionic cells. The animals were bred and housed at the institute for Veterinary Physiology and Biochemistry of the Justus Liebig University Giessen at an ambient temperature of 22.5 °C and air humidity of 50–55% on a 12 h: 12 h light-dark cycle with free access to water and food during the experiment. The animals were anaesthetized with CO2 and killed by exsanguination. The experiments were approved by the named animal welfare officers of the Justus Liebig University (administrative number 577_M) and were performed according to the German and European animal welfare law. 2

European Journal of Pharmacology 864 (2019) 172713

J. Ballout and M. Diener

compartment with 2 × the chamber volume). After a final washing step, carbachol and forskolin were administered as viability control at the end of each experiment. In other experiments, the mucosa-submucosa preparations were preincubated with putative agonists of mast cell receptors such as nicotine, pilocarpine, substance P and norepinephrine, before the effect of compound 48/80 on Isc was tested and compared with a time-dependent control. In the case of substance P, which was dissolved in a 0.1% (w/v) bovine serum albumin (BSA)-containing stock solution, the control tissue was treated with the same volume of 0.1% (w/v) BSA.

2.8. Ca2+ imaging experiments The Ca2+ imaging experiments were carried out at room temperature with the Ca2+ sensitive fluorescent dye fura-2 to measure changes in the cytosolic Ca2+ concentration in a RBL-2H3 monoculture or in cocultures of this mast cell line with intact submucosa or isolated submucosal ganglionic cells, respectively. For this purpose, an inverted microscope (IX-50; Olympus, Hamburg, Germany) endowed with an epifluorescence setup was used, which was connected to a charged coupled device (CCD) camera and a to computer with an image analysis system (Till Photonics, Martinsried, Germany). Cells or tissues were loaded with the membrane permeable form of fura-2, fura-2 acetoxymethylester (fura-2/AM), combined with the nonionic detergent pluronic acid. Loading parameters for the single culture of RBL-2H3 were 6 μmol/l fura-2/AM and 6 mg/l pluronic acid for 90 min, whereas the cells in the coculture were loaded with 3 μmol/l fura-2/AM and 3 mg/l pluronic acid for 1 h. For the coculture of intact submucosa and RBL-2H3 cells, higher concentrations (12 μmol/l fura2/AM and 12 mg/l pluronic acid) and a longer loading period (2 h) was necessary. After washing with Tyrode solution, the coverslips were mounted in the imaging chamber and covered with 1 ml Tyrode solution. The cells were excited alternately with 340 nm or 380 nm and the emission ratio was calculated (340 nm/380 nm) in different regions of interest (ROI), each with the size of one individual cell. Mast cell degranulation induced by compound 48/80 in coculture with enteric neurons leads to an increase in the cytosolic Ca2+ concentration of neighbouring neurons, when the mast cell mediators reach them by diffusion (Bell et al., 2015). Submucosal neurons were identified microscopically according to their specific morphology: a triangular cell body with a different number of processes (a thicker axon and one or more thinner dendrites). To verify the microscopic characterization, we used either KCl as viability control for the isolated neuronal cells to induce a strong depolarization to open voltage-dependent Ca2+ channels resulting in an increase in the fura-2 signal. In case of intact submucosal ganglia, electrical field stimulation was used as functional control. A response to compound 48/80 was accepted by definition when two conditions were fulfilled simultaneously: 1.) the amplitude of the change exceeded the 4-fold standard deviation of the scattering in the fura-2 ratio during the control period just prior to addition of the drug. 2.) The amplitude of the change in the fura-2 ratio exceeded an absolute value of 0.1. The increase in the fura-2 ratio signal induced by membrane depolarization with 30 mmol/l KCl served as viability control for the neurons at the end of each measurement. To elucidate the effects of different agonists on mast cell activation, the monocultured RBL-2H3 cells were preincubated with nicotine, pilocarpine, substance P or norepinephrine, each for 15 min, before they were stimulated with compound 48/80. To test the viability of the monocultured RBL-2H3 cells in the fura-2 experiments, cyclopiazonic acid (10−6 mol/l), a blocker of sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA), was administered at the end of each experiment. For coculture experiments of intact submucosa and RBL-2H3, electrical field stimulation (EFS) was used. A plexiglass holder with two semi-circular platinum wires (with a distance of approximately 12 mm) was put on the imaging chamber. The platinum wires were connected to a pulse stimulator (Grass S44; Warwick, RI, USA), which produced bipolar pulses with a duration of 0.8 ms, a frequency of 50 Hz and a voltage of 40 V over a period of 10 s.

2.5. Culturing of isolated submucosal ganglionic cells After washing and centrifugation (10 s, 1400 g) of the submucosal specimens, the Parsons buffer was removed and replaced with digestion solution consisting of 1.8 mg/ml collagenase type II (273 U/mg; Biochrom, Berlin, Germany) dissolved in Hank's balanced salt solution (without Ca2+ and Mg2+) supplemented with 2.3 mg/ml HEPES and 1.3 mg/ml glucose. The digestion solution was tempered at 37 °C and agitated over a time period of 60–90 min until a turbid and homogenous solution was achieved. During this period, the submucosal specimens were triturated by up and down pipetting of the solution with sterile cannulas in different sizes (21G, 25G and finally 27G). After centrifugation (3 min, 100 g) the supernatant was replaced by NB-A, gently homogenized and centrifuged again (3 min, 100 g). The supernatant was removed and the appropriate amount of NB-A (37 °C) was added, so that 50 μl of this solution could be pipetted per coverslip (diameter 13 mm), which had been coated with poly-L-lysine (0.02 mg/ l). They were transferred into 4-well plates, filled to a final volume of 500 μl per well with NB-A medium and stored up to three days in an incubator (37 °C, 5% (v/v) CO2).

2.6. Culturing of RBL-2H3cells The mast cell equivalent cell line RBL-2H3 was cultivated in MEM α. Twice per week, the cells were passaged by incubating with trypsinEDTA (ethylenediaminetetraacetic acid) for 5 min at 37 °C to separate the cells from the bottom of a Petri dish. An amount of 60,000 cells were transferred into a fresh Petri dish and filled up to 6 ml with MEM α (37 °C). The RBL-2H3 cells were kept in an incubator (37 °C, 5% (v/v) CO2) until passage 30. For Ca2+ imaging experiments, 5000 cells per well were transferred onto poly-L-lysine (0.02 mg/l) coated coverslips into 4-well plates, refilled with 500 μl MEM α and stored in the incubator (37 °C, 5% (v/v) CO2) until the beginning of the experiments.

2.7. Coculture of submucosal ganglionic cells and RBL-2H3 cells The RBL-2H3 and the freshly isolated submucosal ganglionic cells were preincubated separately with different proinflammatory cytokines over a time period of either 1 or 3 days in 4-well plates. TNFα (10 ng/ ml or 100 ng/ml), IL-1β (20 ng/ml) and IFN-γ (100 ng/ml) were added either separately or as cytokine mix to the respective cell medium, which was changed daily. Time-dependent controls were performed in parallel without cytokine exposure. One day before the Ca2+ imaging experiments with the coculture started, RBL-2H3 cells were passaged and 5000 cells per well were transferred onto the cultured isolated submucosal ganglionic cells, refilled with 500 μl MEM α per well and incubated overnight in an incubator (37 °C, 5% (v/v) CO2). RBL-2H3 cells were cocultured with intact submucosa one day after preparation of the submucosa. At this time point, the NB-A was removed and 5000 RBL-2H3 cells were added to each well. The coculture was covered with 500 μl MEM α and kept for further 15 min in the incubator (37 °C, 5% (v/v) CO2).

2.9. Immunocytochemistry The RBL-2H3 cells, which were mounted on poly-L-lysine (0.02 mg/ l) coated coverslips and incubated over a period of 3 days with and without the cytokine mix, were rinsed twice with PBS for 5 min and fixed with PBS containing 4% (w/v) paraformaldehyde for at least 15 min at 4 °C. After washing with PBS, the cells were incubated for 1 h at room temperature in a blocking solution containing PBS with 3

European Journal of Pharmacology 864 (2019) 172713

J. Ballout and M. Diener

Table 1 Effect of TNFα on basal and secretagogue-induced Isc.

μEq/h/cm + TNFα (10 ng/ml) − TNFα (10 ng/ml) + TNFα (50 ng/ml) − TNFα (50 ng/ml) + TNFα (100 ng/ml) − TNFα (100 ng/ml)

Baseline Isc

ΔIsc induced by compound 48/80

1.96 1.59 1.99 1.21 2.46 1.30

1.73 1.31 2.03 1.67 1.25 1.05

ΔIsc induced by histamine

ΔIsc induced by carbachol

ΔIsc induced by forskolin

9.88 ± 0.38b 9.55 ± 0.76b 11.07 ± 0.64b 9.51 ± 0.64b 9.02 ± 0.46b 9.55 ± 0.57b

9.88 ± 0.78b 9.51 ± 1.02b 11.09 ± 0.99b 9.72 ± 0.45b 8.96 ± 0.79b 9.91 ± 1.04b

2.63 1.99 2.22 2.37 1.85 2.33

2

± ± ± ± ± ±

0.40 0.47 0.45 0.52 0.31a 0.45

± ± ± ± ± ±

0.29b 0.36b 0.45b 0.43b 0.21b 0.22b

± ± ± ± ± ±

0.76b 0.27b 0.27b 0.28b 0.19b 0.34b

Effect of 1 h preincubation with different concentrations of TNFα (at the serosal side) on baseline Isc or changes in Isc induced by compound 48/80 (15 μg/ml at the serosal side), histamine (10−4 mol/l at the serosal side), carbachol (5·10−5 mol/l at the serosal side), and forskolin (5·10−6 mol/l at mucosal and serosal side). The effect of secretagogues is given as difference to the baseline (ΔIsc) just prior administration of the respective agonist. Data expressed as mean ± S.E.M., n = 9–11. a P < 0.05 compared to respective time-dependent control (unpaired t-test), b P < 0.05 versus baseline (paired t-test).

conditions in vitro, in a first step the effects of different proinflammatory cytokines on baseline parameters were tested in Ussing chamber experiments. As typical proinflammatory cytokines, whose concentrations are elevated in the gut wall in different models of inflammatory bowel diseases, TNFα (Neurath et al., 1997), IL-1β (Bauer et al., 2010), and IFN-γ (Pérez-Navarro et al., 2005) were selected. Especially a mix of the three (or two of these) cytokines was able to modify intestinal ion transport via the epithelial Na+ channel (ENaC) in vitro (Barmeyer et al., 2004). In the case of the pleiotropic cytokine TNFα, which can e.g. enhance both cell survival as well as death of cells via apoptosis (Gupta, 2002), different concentrations were tested (10, 50 and 100 ng/ml at the serosal side). For the other two proinflammatory cytokines, i.e. IL-1β (20 ng/ml at the serosal side) and IFN-γ (100 ng/ml at the serosal side), effective concentrations from the literature (Barmeyer et al., 2004) were selected. TNFα, in the highest concentration tested (100 ng/ml at the serosal side), induced a significant increase of basal short-circuit current (Isc) within 1 h after administration (Table 1, Fig. 1A). In parallel to baseline Isc, TNFα induced an increase in basal tissue conductance (Gt), which reached statistical significance for concentrations ≥ 50 ng/ml (Table 2). IL-1β (20 ng/ml at the serosal side) did not change the basal Isc (Fig. 1A), but caused a significant increase in baseline Gt (Table 2), whereas INF-γ (100 ng/ml at the serosal side) did not affect basal electrical properties of the mucosa (Fig. 1A, Table 2). Mast cell degranulation was evoked by compound 48/80 (15 μg/ml; corresponds 10−4 mol/l with reference to the monomer) and the resulting anion secretion was measured with and without prior treatment with these cytokines. None of the cytokines, when administered alone, was able to modify the increase in Isc or Gt evoked by compound 48/80 (Fig. 1B, Table 2). One of the main prosecretory mediators of mast cells is histamine, which induces an intestinal anion secretion by activation of histamine receptors on the epithelium (Schultheiss et al., 2006) and on secretomotor submucosal neurons (Bell et al., 2015). The Isc as well as the increase in Gt induced by histamine (10−4 mol/l at the serosal side) was significantly attenuated after preincubation with IL-1β (20 ng/ml at the serosal side), whereas none of the other cytokines, when administered alone, affected the response to histamine on Isc or Gt (Fig. 1C, Table 2). In order to find out whether the cytokines might influence the overall secretory capacity of the epithelium, carbachol (5·10−5 mol/l at the serosal side) and forskolin (5·10−6 mol/l at the mucosal and the serosal side), i.e. a Ca2+- and a cyclic adenosine monophosphate (cAMP)-dependent secretatogue, were administered at the end of each experimental series. The response to carbachol was significantly reduced after preincubation with IL-1β (Fig. 1D) suggesting an impairment of epithelial Ca2+-dependent Cl− secretion. However, this inhibition was not mimicked by the other two cytokines (Fig. 1D) and none of the individually applied cytokines modified the secretion induced by the cAMP-dependent secretagogue forskolin (Fig. 1E).

0.05% (v/v) triton-X-100 and 10% (v/v) donkey serum. This was followed by incubation with the primary antibody against activated caspase-3 (Cas3) overnight at 4 °C. The antibody was dissolved in the blocking solution in a dilution of 1:2000. In parallel, negative controls without the primary antibody were performed. These negative controls did not result in any staining (data not shown). After repeated washing steps, the cells were loaded with the secondary antibody Cy3 donkey anti-rabbit (1:1000) dissolved in PBS containing 0.05% triton-X-100 (v/v) for 2 h at room temperature and were rinsed again. For nuclear staining the sections were incubated with 4,6-diamidio-2-phenylindoldilactate (DAPI, 3·10−7 mol/l) for 5 min and embedded with Hydromount® (Biozym, Oldendorf, Germany). Pictures were taken with the fluorescence microscope (Nikon 80i; Nikon, Düsseldorf, Germany). For each coverslip five randomly pictures were taken from both groups and mast cells were counted in a blinded fashion using NIS Elements 2.30 software (Nikon) using a moderate background correction. 2.10. Drugs Carbachol, compound 48/80 (condensation product of methoxy-Nmethylphenethylamine and formaldehyde; polymer consisting of 3–6 monomers), nicotine hydrogen tartrate, norepinephrine bitartrate, and pilocarpine hydrochloride were dissolved in aqueous stock solutions. Substance P was dissolved in 0.1% (w/v) BSA solution. Forskolin was dissolved in 96% (v/v) ethanol. Cyclopiazonic acid was dissolved in dimethyl sulfoxide (DMSO). If not indicated differently, drugs were from Sigma, Taufkirchen, Germany. 2.11. Statistics Results are given as mean ± standard error of the mean (S.E.M.) with number (n) of investigated tissues or cells. For the imaging experiments, cultures from at least three different animals were used for each experimental series. To compare two groups, an unpaired t-test or a Mann-Whitney-U-test was performed. In order to find out, which test method had to be used, F-test was applied. Paired t-test was used to compare the change of a parameter induced by a drug in comparison to the baseline just prior drug administration of the same cell or tissue. To compare the proportion of responding cells in control and experimental group, a χ2-test was performed. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Impact of individual proinflammatory cytokines on intestinal secretion and permeability As the main intention of this study was to find out whether mast cell – neuron interactions in the gut might be altered under inflammatory 4

European Journal of Pharmacology 864 (2019) 172713

J. Ballout and M. Diener

Fig. 1. Effect of preincubation with individual cytokines for 1 h (black bars) or a cytokine mix (shaded bars) for different time intervals on baseline Isc (measured 5 min before administration of compound 48/80; A) or the Isc induced by compound 48/80 (15 μg/ml at the serosal side; B), histamine (10−4 mol/l at the serosal side; C), carbachol (5·10−5 mol/l at the serosal side; D), and forskolin (5·10−6 mol/l at mucosal and serosal side; E) in comparison to time-dependent controls (white bars). The effect of secretagogues is given as difference to the baseline (ΔΙsc) just prior administration of the respective agonist. Concentrations of the cytokines (applied either alone or in the cytokine mix) were: TNFα (100 ng/ml at the serosal side), IL-1β (20 ng/ml at the serosal side) and IFN-γ (100 ng/ml at the serosal side). Data expressed as mean + S.E.M., n = 5–10. *P < 0.05 versus the respective control group (unpaired t-test).

the cytokine mix) the secretory responses evoked by compound 48/80, histamine, carbachol and forskolin were tested. At these time points, neither basal Isc (Fig. 1A) nor basal Gt (Table 2), measured 5 min before administration of compound 48/80, differed from the respective timedependent control series. After 1 h pretreatment with the cytokine mix, the increase in Isc (ΔIsc) induced by compound 48/80 (15 μg/ml at the serosal side) tended to be reduced compared to the respective control series, but this difference did not reach statistical significance (Fig. 1B). The Isc induced by histamine (10−4 mol/l at the serosal side), however, was significantly diminished when the tissues had been incubated for 1 h with the cytokine cocktail (Fig. 1C). The same held for the Isc evoked by carbachol (5·10−5 mol/l at the serosal side), which was significantly smaller compared to the time-dependent control series, when the tissue had been pretreated for 1 or 2 h with the cytokine combination (Fig. 1D). The response to the cAMP-dependent secretagogue forskolin (5·10−6 mol/l at the mucosal and the serosal side) was also inhibited 2 h after administration of the cytokine cocktail (Fig. 1E). Three hours after administration of the cytokine mix, none of the measured parameters was changed any more in comparison to the timedependent control series (Fig. 1A–E).

3.2. Effect of a cytokine cocktail on intestinal secretion and permeability Under in vivo conditions usually not only individual cytokines are upregulated. Instead, the relevant players involved in regulation of epithelial secretion, i.e. mast cells, enteric neurons and enterocytes will be exposed to combinations of different proinflammatory cytokines. Therefore, in the subsequent experiments, a mix of the three individually tested cytokines (100 ng/ml TNFα, 20 ng/ml IL-1β and 100 ng/ml IFN-γ) was administered at the serosal side of distal colon. Overall, three experimental series were performed, in which the tissues were pretreated for 1, 2 or 3 h with this cocktail, before other secretagogues were administered. The cytokine mix caused a transient increase of Isc (Fig. 2): when all experimental series were pooled (n = 19) and compared to time-dependent control experiments (n = 24), the cytokine cocktail induced a significant increase in Isc of 1.08 ± 0.19 μEq/h/cm2 (n = 19; P < 0.05 versus time-dependent control, unpaired t-test). In parallel, they led to a significant increase in Gt, which amounted to 0.76 ± 0.23 mS/cm2 (n = 19; P < 0.05 versus time-dependent control, U test) within 30 min after administration of the cytokine cocktail. At three different time points (1, 2 and 3 h after administration of 5

European Journal of Pharmacology 864 (2019) 172713

J. Ballout and M. Diener

Table 2 Effect of single cytokines and cytokine mix on basal and secretagogue-induced changes in Gt. Baseline Gt

ΔGt induced by compound 48/80

ΔGt induced by histamine

ΔGt induced by carbachol

ΔGt induced by forskolin

9.88 ± 0.67 10.43 ± 0.67 13.21 ± 1.14a 9.88 ± 0.83 14.50 ± 1.66a 10.52 ± 0.46 12.00 ± 0.64a 9.45 ± 0.20 11.50 ± 1.06 11.01 ± 0.95

0.72 0.68 1.01 0.57 0.68 0.50 0.39 0.48 0.92 0.67

± ± ± ± ± ± ± ± ± ±

0.16b 0.29b 0.13b 0.16b 0.23b 0.25b 0.13b 0.12b 0.43b 0.22b

4.43 4.61 5.07 4.48 4.07 4.29 3.21 4.75 3.87 4.36

± ± ± ± ± ± ± ± ± ±

0.26b 0.45b 0.35b 0.45b 0.34b 0.38b 0.34a,b 0.57b 0.40b 0.47b

6.42 6.12 7.15 5.73 5.69 5.97 4.77 6.42 5.12 5.74

± ± ± ± ± ± ± ± ± ±

0.62b 0.88b 0.73b 0.36b 0.66b 0.82b 0.63b 0.96b 0.54b 0.87b

3.06 2.76 3.34 2.88 3.07 2.76 2.78 2.37 2.15 2.18

± ± ± ± ± ± ± ± ± ±

0.43b 0.48b 0.50b 0.27b 0.34b 0.42b 0.51b 0.33b 0.26b 0.23b

11.68 ± 1.39 10.87 ± 0.54 10.41 ± 0.90 10.32 ± 1.02 9.15 ± 0.58 7.85 ± 1.15

0.70 1.18 0.30 0.67 1.76 0.71

± ± ± ± ± ±

0.20b 0.40b 0.09b 0.18b 0.68b 0.19b

3.93 5.15 3.10 4.11 3.40 3.41

± ± ± ± ± ±

0.65b 0.39b 0.32b 0.38b 0.50b 0.30b

4.35 6.75 3.75 5.23 5.38 4.87

± ± ± ± ± ±

0.67a,b 0.71b 0.39b 0.70b 0.87b 0.49b

3.21 3.16 2.12 2.99 3.71 3.03

± ± ± ± ± ±

0.61b 0.69b 0.33b 0.50b 0.73b 0.39b

2

mS/cm

Single cytokines + TNFα (10 ng/ml) − TNFα (10 ng/ml) + TNFα (50 ng/ml) − TNFα (50 ng/ml) + TNFα (100 ng/ml) − TNFα (100 ng/ml) + IL-1β (20 ng/ml) − IL-1β (20 ng/ml) + IFN-γ (100 ng/ml) − IFN-γ (100 ng/ml) Cytokine mix + Cytokine mix (1 h) − Cytokine mix (1 h) + Cytokine mix (2 h) − Cytokine mix (2 h) + Cytokine mix (3 h) − Cytokine mix (3 h)

Effect of preincubation with individual cytokines for 1 h or preincubation with a cytokine mix for 1–3 h on baseline Gt or changes in Gt induced by compound 48/80 (15 μg/ml at the serosal side), histamine (10−4 mol/l at the serosal side), carbachol (5·10−5 mol/l at the serosal side), and forskolin (5·10−6 mol/l at mucosal and serosal side). The effect of secretagogues is given as difference to the baseline (ΔGt) just prior administration of the respective agonist. Data expressed as mean ± S.E.M., n = 5–11. a P < 0.05 compared to respective time-dependent control (unpaired t-test or U test), b P < 0.05 versus baseline (paired t-test).

cells of interest to cytokines, which is closer to the in vivo situation during inflammatory conditions. In this coculture, an increase in the cytosolic Ca2+ concentration (measured as increase in the fura-2 ratio signal) in neurons after induction of mast cell degranulation with compound 48/80 can be taken as readout for neuronal activation by mast cell mediators (Bell et al., 2015). Exposure of the coculture to compound 48/80 (15 μg/ml) causes a strong increase in the cytosolic Ca2+ concentration of the neurons within this coculture. Not all submucosal neurons respond to mast cell degranulation. When all control experiments were pooled, in 368 out of 561 (69%) neurons an increase of the fura-2 ratio signal was evoked by mast cell degranulation. This percentage is in accordance with a previous study from our group (Bell et al., 2015) and probably reflects differences in receptor expression and excitability of the different types of neurons present in this part of the enteric nervous system. When TNFα was used in a low concentration (10 ng/ml), the amplitude of the fura-2 ratio signal was significantly increased in comparison to an untreated control group (Fig. 3), whereas the percentage of neurons responding to the mast cell degranulation remained unchanged (Fig. 4). A different pattern was observed, when the TNFα

3.3. Modulation of the response of submucosal neurons to mast cell mediators by cytokines The reduction of the secretory effect of mast cell degranulation with compound 48/80 after preincubation with the cytokine mix (Fig. 1B) might be due to an impairment of the secretory capacity of the epithelium as shown by the reduced responses to carbachol and forskolin (Fig. 1D and E). However, also enteric neurons mediate the secretion induced by mast cell mediators as shown by experiments, in which the Isc induced by antigen contact of tissues from sensitized animals is strongly inhibited by the neurotoxin tetrodotoxin (Javed et al., 1992; Hug et al., 1996). Consequently, it might be thought that in addition the stimulation of secretomotor neurons by mast cell mediators might be reduced by cytokines. To test this hypothesis, a coculture of isolated submucosal ganglionic cells and the mast cell equivalent cell line RBL2H3 was incubated with individual proinflammatory cytokines or a mix of them in comparison to a time-dependent control group. In contrast to the Ussing chamber experiments, in which – due to the limited viability of the ex vivo preparations – only acute, i.e. short-term effects of cytokines can be tested, this model allows the prolonged exposure of the

Fig. 2. Time course of the short-circuit current (Isc) induced by serosal administration of a cytokine mix (100 ng/ml TNFα, 20 ng/ml IL-1β, and 100 ng/ml IFN-γ; A) in comparison to a time-dependent control (B). Values are means (black line) ± S.E.M. (grey area), n = 8–9. 6

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Fig. 3. Increase in the fura-2 ratio signal induced by the mast cell activator compound 48/80 (15 μg/ml) in isolated submucosal neurons cocultured with the mast cell line RBL-2H3. The effect of compound 48/ 80 was tested with (black bars) and without (white bars) preincubation with individual cytokines for 1 d or with (shaded bars) and without (white bars) preincubation with a cytokine mix for 1–3 d. Concentrations of the cytokines in the cytokine mix were: TNFα (100 ng/ml), IL-1β (20 ng/ml) and IFNγ (100 ng/ml). Data expressed as mean + S.E.M. and are given as difference (Δfura-2 ratio) to the baseline just prior administration of compound 48/ 80. n = 32–166 tested cells from at least three different animals; *P < 0.05 (unpaired t-test or U test) compared to the respective control.

decrease in mast cell density in animals sensitized against egg albumin, when in addition to the sensitization a colitis was induced by luminal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Becker et al., 2019). Thus, we asked the question whether survival of mast cells might be reduced in an inflammatory environment. Therefore, cultured RBL-2H3 cells were treated over 3 d with a cytokine cocktail (100 ng/ ml TNFα, 20 ng/ml IL-1β, and 100 ng/ml IFN-γ). Incubation of RBL2H3 with the cytokine mix over a period of three days caused an obvious loss of cell density. To elucidate if this is caused by apoptosis or necrosis, the mast cells were stained with an antibody against the active form of caspase 3 (Fig. 5), which is one of the key enzymes activated during apoptosis. There was a significant increase of Cas3 positive cells after preincubation with the cytokine mix over a period of 3 days (44%, n = 1070 apoptotic cells out of 2461 cells counted) compared to control cells (9%, n = 256 apoptotic cells out of 2902 cells counted; P < 0.05, χ2-test).

concentration was elevated to 100 ng/ml. In this case the percentage of neurons responding during the mast cell degranulation evoked by compound 48/80 increased to 90% (46 out of 51 neurons) in comparison to 53% (42 out of 80 neurons) in the respective control series (P < 0.05, χ2-test; Fig. 4), whereas the mean amplitude of the rise in the fura-2 signal in the responding cells remained constant (Fig. 3). Neither pretreatment (for 1 d) with IL-1β (20 ng/ml) nor with IFN-γ (100 ng/ml) had any significant effect on the amplitude of the neuronal response during exposure to compound 48/80 (Fig. 3) nor on the percentage of responding neurons (Fig. 4). Surprisingly, when added in combination as cytokine mix for 1 d (100 ng/ml TNFα, 20 ng/ml IL-1β, 100 ng/ml IFN-γ), the amplitude of the neuronal fura-2 response was significantly reduced by about 40% from 2.15 ± 0.23 for control group (n = 48) to 1.34 ± 0.15 in the cytokine-pretreated group (n = 68; P < 0.05; unpaired t-test; Fig. 3). When the exposure to the cytokine cocktail was prolonged to 3 d, this inhibition vanished and a significantly higher percentage of neurons (86% versus 66% under control conditions; P < 0.05, χ2-test) responded with a Ca2+ signal during mast cell degranulation induced by compound 48/80 (Fig. 4).

3.5. Mast cell-mediated anion secretion is altered by agonists of neurotransmitter receptors The communication between mast cells and enteric neurons is thought to function in a bidirectional way. Mast cell mediators stimulate enteric neurons (see e.g. Schemann et al., 2005), but vice versa enteric neurons themselves have been shown to induce mast cell

3.4. A proinflammatory cytokine mix induces apoptosis in RBL-2H3 cells In a recent in vivo study, we observed an unexpected numeric

Fig. 4. Percentage of submucosal neurons responding to mast cell activation with compound 48/ 80 (15 μg/ml) after preincubation with individual cytokines (black bars) or a cytokine mix (shaded bars) compared to the respective control series without cytokine pretreatment (white bars). Concentrations of the cytokines in the cytokine mix were: TNFα (100 ng/ml), IL-1β (20 ng/ml) and IFNγ (100 ng/ml). In each column the total number of neuronal responder are written compared to all neurons reacting to KCl (30 mmol/l) as viability control. *P < 0.05 compared to the respective control (χ2-test).

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Fig. 5. Immunocytochemical staining of RBL-2H3 cells with a primary antibody against activated caspase 3 (Cas3; 1st column) and nuclear staining with DAPI (2nd column) after 3 day preincubation with a cytokine mix (1st row) in parallel to a timedependent control without cytokine mix (2nd row). The 3rd column gives an overlay of the respective pictures, whereas the 4th column shows a larger magnification of the overlay picture of an independent experiment. Concentrations of the cytokines in the cytokine mix were: TNFα (100 ng/ml), IL-1β (20 ng/ml), and IFN-γ (100 ng/ml). White calibration car: 50 μm; yellow calibration bar: 10 μm. Representative staining of three independent experiments.

Table 3 Effect of different agonists on Isc in Ussing chamber experiments. Agonist μEq/h/cm

ΔIsc induced by agonist

ΔIsc induced by carbachol

ΔIsc induced by forskolin

0.12 0.16 0.90 0.03 0.22 0.08 3.43 0.05 6.95 0.12

± ± ± ± ± ± ± ± ± ±

0.07 0.07 0.33a,b 0.05 0.06b 0.02 1.11a,b 0.03 0.93a,b 0.02

10.32 ± 1.10b 11.75 ± 1.04b 10.98 ± 0.97b 10.62 ± 1.24b 12.62 ± 1.24b 10.20 ± 1.09b 10.73 ± 1.78b 9.27 ± 1.19b 6.11 ± 1.71b 6.12 ± 1.12b

3.44 3.74 3.33 3.18 3.00 1.82 3.61 2.83 2.58 2.78

± ± ± ± ± ± ± ± ± ±

0.64b 0.33b 0.55b 0.42b 0.40a,b 0.22b 0.69b 0.43b 0.21b 0.48b

0.20 0.21 1.32 0.14 3.97 0.12

± ± ± ± ± ±

0.06b 0.06 0.25a,b 0.07 0.84a,b 0.04

12.01 ± 1.88b 11.42 ± 1.58b 8.45 ± 1.53b 7.05 ± 1.23b 10.27 ± 2.10b 9.67 ± 1.91b

4.38 3.79 3.43 2.90 2.93 2.64

± ± ± ± ± ±

0.80b 0.42b 0.25b 0.51b 0.58b 0.55b

9.44 ± 1.30b 10.14 ± 1.43b

2.72 ± 0.49b 3.02 ± 0.46b

2

Cholinergic agonists + Nicotine (10−7 mol/l) − Nicotine (10−7 mol/l) + Nicotine (10−6 mol/l) − Nicotine (10−6 mol/l) + Pilocarpine (10−6 mol/l) − Pilocarpine (10−6 mol/l) + Pilocarpine (10−5 mol/l) − Pilocarpine (10−5 mol/l) + Pilocarpine (5·10−5 mol/l) − Pilocarpine (5·10−5 mol/l) Neurokinine + Substance P (10−11 mol/l) − Substance P (10−11 mol/l) + Substance P (10−10 mol/l) − Substance P (10−10 mol/l) + Substance P (10−9 mol/l) − Substance P (10−9 mol/l) Adrenergic agonist + Norepinephrine (5·10−6 mol/l) − Norepinephrine (5·10−6 mol/l)

−0.73 ± 0.24a −0.12 ± 0.03

Effect of different agonists on Isc (2nd column) and the Isc induced after 5 min preincubation with the respective agonist by carbachol (5·10−5 mol/l at the serosal side; 3rd column) and forskolin (5·10−6 mol/l at mucosal and serosal side; 4th column). For nicotine (10−7 mol/l) the preincubation time was prolonged to 15 min. The effect of secretagogues is given as difference to the baseline (ΔIsc) just prior administration of the respective agonist. Data expressed as mean ± S.E.M., n = 4–9. a P < 0.05 compared to respective time-dependent control (unpaired t-test or U test), b P < 0.05 versus baseline (paired t-test). Fig. 6. Effect of compound 48/80 (15 μg/ml at the serosal side) on Isc in the presence (grey bars) or absence (white bars) of different agonists. The agonists nicotine (10−6 mol/l), pilocarpine (10−6 mol/l, 10−5 mol/l, 5·10−5 mol/l), substance P (10−11 mol/l, 10−10 mol/l, 10−9 mol/l) or norepinephrine (5·10−6 mol/l) were administered on the serosal side; preincubation time was in general 5 min, except for nicotine (10−7 mol/l), where preincubation time amounted to 15 min. Data expressed are given as difference of the compound 48/ 80-induced current to the baseline just prior administration of the mast cell degranulator (ΔIsc) and are means + S.E.M., n = 4–9. *P < 0.05 versus response of compound 48/80 in the absence of the respective agonist (unpaired t-test or U test).

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degranulation by neuropeptides such as substance P (Buhner and Schemann, 2012). Furthermore, muscarinic (Bani-Sacchi et al., 1986) and adrenergic (Butchers et al., 1991) receptors have been reported to stimulate degranulation of mast cells, whereas nicotinic receptors exert an inhibitory action (Kageyama-Yahara et al., 2008). Thus we tested in Ussing chamber experiments whether pretreatment with agonists of these receptors alter the Isc induced by the mast cell stimulator compound 48/80. Nicotine in a concentration of 10−6 mol/l (at the serosal side) induced a small increase in Isc of 0.90 ± 0.33 μEq/h/cm2 (n = 6; P < 0.05; paired t-test; Table 3). When subsequently compound 48/80 (15 μg/ml at the serosal side) was administered, the increase of Isc induced by the mast cell degranulator was strongly inhibited (0.28 ± 0.13 μEq/h/cm2 in the presence of nicotine compared to 1.57 ± 0.42 μEq/h/cm2 in the absence of nicotine, n = 6, P < 0.05; U test; Fig. 6). In contrast to the in vivo situation, where vagal nerve stimulation stimulates histamine release from rat ileum in an atropine-sensitive manner (Bani-Sacchi et al., 1986), pretreatment with pilocarpine (10−6 mol/l - 5·10−5 mol/l at the serosal side) did not affect significantly the Isc induced by compound 48/80 (Fig. 6), although pilocarpine itself led to a significant increase of Isc in a concentration-dependent manner (Table 3). Substance P (10−11 mol/l - 10−9 mol/l at the serosal side) caused a concentration-dependent increase in Isc (Table 3), too, but did not alter the response to compound 48/80 (Fig. 6). The catecholamine norepinephrine (5·10−6 mol/l at the serosal side) induced a negative Isc (Table 3) reflecting K+ secretion (Hörger et al., 1998) without affecting the increase in Isc induced by the subsequent administration of compound 48/80 (Fig. 6).

Fig. 7. Electrical field stimulation (EFS with 40 V, 0.8 ms, 50 Hz, 10 s) evokes an instantaneous activation of a submucosal neuron followed by a delayed response of a neighbouring mast cell (represented as original tracing of fura-2 ratio). Intact submucosa from rat distal colon was cocultured with the mast cell equivalent cell line RBL-2H3. Overall, 33 out of 58 mast cells (from n = 16 different wells with ganglia from N = 11 different animals) showed a similar response, whereas in the same preparations 94 out of 94 tested neurons responded to EFS with an increase of the fura-2 ratio. Table 4 Ca2+ responses of RBL-2H3 to different agonists. Agonist

3.6. Effect of stimulation of neurotransmitter receptors on cytosolic Ca2+ concentration of RBL-2H3 cells

Nicotine (10−6 mol/l) Pilocarpine (10−6 mol/l) Substance P (10−9 mol/l) Norepinephrine (5·10−6 mol/l)

An increase in the cytosolic Ca2+ concentration of the mast cells is centrally involved in the process of mast cell mediator release (Oda et al., 2013). Consequently, in coculture experiments, where RBL-2H3 cells can easily be distinguished by their morphology, their activation can be monitored by fura-2 imaging. In order to test whether they respond to neurotransmitters released by enteric neurons, in Ca2+ imaging experiments neurons from submucosal ganglia were stimulated by electrical field stimulation (EFS). Indeed, a fraction of mast cells (33 out of 58 cells tested in n = 16 different wells with ganglia from N = 11 different animals), neighboured to submucosal ganglia responded with a delayed increase of the cytosolic Ca2+ concentration during neuronal stimulation (Fig. 7). This increase was not only delayed, but also smaller in the amplitude (Δfura-2 ratio 0.14 ± 0.02, n = 33) in comparison to the response of the neurons (Δfura-2 ratio 0.47 ± 0.03, n = 94). In order to study the potential effect of agonists acting on neurotransmitter receptors expressed by mast cells more directly, RBL-2H3 cells (in monoculture) were exposed to the agonists tested in the Ussing chamber experiments described above. All agonists tested, i.e. nicotine (10−6 mol/l), pilocarpine (10−6 mol/l), norepinephrine (5·10−6 mol/l), and substance P (10−9 mol/l) induced an increase in the fura-2 ratio signal in a fraction of the RBL-2H3 cells (Table 4). The highest number of responders (53%) was observed for substance P, which is in accordance to the known degranulating effect of this neuropeptide (Buhner and Schemann, 2012). However, nicotine also evoked - albeit much weaker - an unexpected increase in the cytosolic Ca2+ concentration in 22% of the RBL-2H3 cells (Table 4).

Δ fura-2 ratio

0.67 0.83 1.18 1.28

± ± ± ±

0.12a 0.12a 0.14a 0.27a

Responder n

%

15/68 15/63 29/55 23/64

22 23 53 36

Effect of different agonists on the cytosolic Ca2+ concentration of RBL-2H3 cells. Given is the increase of the fura-2 ratio signal in the responding cells compared to the baseline just prior administration of the agonist (Δfura-2 ratio; 2nd column), the number of responding cells in comparison of the total number of cells tested (3rd column) and the percentage of the responding cells (4th column). Cyclopiazonic acid (10−6 mol/l), when administered as viability control at the end of each experiment, induced an increase in the fura-2 ratio of 2.71 ± 0.13 (pooled data, n = 250). Data expressed as mean ± S.E.M., n = 55–68, from three independent experiments. a P < 0.05 versus baseline (paired t-test).

Initially, the monocultured cells were treated with the same concentration of compound 48/80 (15 μg/ml) as used in the Ussing chamber or the coculture experiments. However, this concentration evoked such a large change of the fura-2 ratio signal in all cells measured (increase in the fluorescence evoked by 340 nm and decrease in fluorescence evoked by 380 nm) that the 380 nm signal approached baseline in most cells. Consequently, the concentration of compound 48/80 was lowered to 1.5 μg/ml for this series of experiments, which resulted in lower fura-2 responses (Fig. 8), but also in the fact that not all RBL-2H3 cells in the culture responded to the agonist at this obvious near threshold concentration (Fig. 9). However, the use of such as ‘near threshold’ concentration evoking submaximal Ca2+ responses was a necessary prerequisite to create conditions allowing to observe not only inhibition of mast cell responses, but also putative enhancement after treatment with agonists. Pretreatment with all four agonists for 15 min reduced the response of the RBL-2H3 cells to compound 48/80. The strongest reduction in the amplitude of fura-2 signal (> 60%) was observed after pretreatment with nicotine (10−6 mol/l; Fig. 8), which would be in accordance with the known inhibitory effect of nicotinic receptor stimulation on mast cell function (Kageyama-Yahara et al., 2008). Paradoxically (see

3.7. Activation of neurotransmitter receptors on RBL-2H3 cells modify their response to compound 48/80 In a final step we investigated whether pretreatment with the agonists, as described above, modifies the response of the mast cell equivalent RBL-2H3 cells to the mast cell stimulator compound 48/80. 9

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Fig. 8. Increase of the fura-2 ratio signal (Δfura-2 ratio) in RBL-2H3 cells induced by compound 48/80 (1.5 μg/ml) in the presence (black bars) or absence (white bars) of different agonists. Cells were preincubated for 15 min with nicotine (10−6 mol/l), pilocarpine (10−6 mol/l), substance P (10−9 mol/l) or norepinephrine (5·10−6 mol/l) before compound 48/80 was administered. Data expressed as mean + S.E.M., n = 55–68 cells. *P < 0.05 versus response to compound 48/80 in the absence of the respective agonist (unpaired t-test or U test).

is the reason of the secretory diarrhea or just a consequence to the inflammation, is still under debate (König et al., 2016). Different studies revealed that proinflammatory cytokines, especially TNFα and IFN-γ, affect the epithelial barrier function by altering the cytoskeleton or tight junction expression (Salim and Söderholm, 2011; Wittkopf et al., 2014). Electrophysiologically, this leads to an increase in tissue conductance (Gt) as shown e.g. in the human colonic tumor cell line HT29/ B6, where preincubation with TNFα (100 ng/ml) decreases tissue resistance by 80% (Schmitz et al., 1999). This was confirmed in the present experiments, where the individual cytokines TNFα (50 or 100 ng/ml) and IL-1β or a mix of proinflammatory cytokines elevated the basal Gt significantly with a maximum after 30 min (Table 2). This response was paralleled by an increase in Isc suggesting an enhanced anion secretion (Table 1, Figs. 1 and 2). Similar effects have been observed in specimens from patients with IBD, where basal Isc and Gt were significantly increased (Crowe et al., 1997). In patients with IBD, elevated serum levels of different proinflammatory cytokines, such as TNFα, IL-1β and IFN-γ, were detected and seem to play an essential role in the signaling pathways leading to secretory diarrhea during IBD (Bauer et al., 2010; Singh et al., 2016). These cytokines are known to exert prosecretory effects on intestinal epithelial cells (Sanchez de Medina et al., 2002). For example, in human distal colon TNFα and IL-1β, but not IFN-γ, evoke an increase of Isc reflecting enhanced anion secretion (Schmitz et al., 1996; Bode et al., 1998). For a long time it was thought that active anion secretion induced by prosecretory cytokines underlies the diarrhea in patients with

Discussion), a reduction of similar amplitude was observed after preincubation with substance P (10−9 mol/l; Fig. 8). This was concomitant with a significant decrease in the percentage of RBL-2H3 cells responding to compound 48/80 after pretreatment with nicotine or substance P (Fig. 9). The smallest reduction of Δfura-2 ratio to compound 48/80 was found when the mast cells were pretreated with pilocarpine (10−6 mol/l; 0.67 ± 0.11, n = 58 compared to control 1.15 ± 0.10, n = 51; P < 0.05; Fig. 8), which did not affect the percentage of responding mast cells (Fig. 9). Even norepinephrine (5·10−6 mol/l) had only a marginal effect on the percentage of RBL-2H3 cells responding to compound 48/80 (Fig. 9), but reduced significantly the amplitude of the induced fura-2 signal (Fig. 8). None of the RBL-2H3 cells pretreated with one of these agonists showed a diminished response to the viability control cyclopiazonic acid (10−6 mol/l; data not shown).

4. Discussion 4.1. Changes in epithelial functions after in vitro induction of inflammation In the pathogenesis of inflammatory bowel diseases (IBD) disturbances in the complex interplay between the epithelium, the enteric nervous system, the immune system and the intestinal microbiom lead to a chronic inflammation in the intestine (Bernardazzi et al., 2016). One of the central pathophysiologic mechanisms involved in IBD is an impaired epithelial barrier function, which leads to a passive flow of ions and water into the gut lumen. If a damage of the epithelial barrier

Fig. 9. Percentage of RBL-2H3 responding to compound 48/80 (1.5 μg/ml) after preincubation with different agonists (black bars) compared to untreated controls (white bars). Agonist concentrations were: nicotine (10−6 mol/l), pilocarpine (10−6 mol/l), substance P (10−9 mol/l), or norepinephrine (5·10−6 mol/l); preincubation time was 15 min. In each column the total number of responders to compound 48/80 are written compared to all cells reacting to cyclopiazonic acid (10−6 mol/l) as viability control. *P < 0.05 compared to the respective control (χ2-test).

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secretion induced by compound 48/80 was reduced by 1 h pretreatment with the cytokine mix (Fig. 1B). This drug degranulates mast cells present in the intestinal wall of the mucosa-submucosa preparation used. This reduction fits well to a previous study from our lab, where we observed that the antigen-induced anion secretion in specimens from sensitized animals in Ussing chamber experiments was reduced, when in addition a colitis had been induced (Becker et al., 2019). As this was not only observed in the colon (where an inflammation had been induced by topical administration of TNBS), but also in the jejunum, i.e. distant from the inflamed area, a systemic effect had been speculated, e.g. induced by circulating cytokines. The reason for this downregulation is by now unclear. Some studies reported that mast cell density and histamine levels in IBD patients and animals with experimental colitis are increased (He, 2004; Rijnierse et al., 2007). Other data contradict this upregulation as in tissue samples of patients with IBD a reduced mast cells density was observed (Bischoff et al., 1996b). In our previous study we observed a trend for a lower density of mast cells in the lamina propria in the colitis group compared to tissues from untreated rats, where the mast cell number amounted to 10.2 ± 1.0 per mm2 in mucosa-submucosa preparations of distal colon (Becker et al., 2019). One explanation can be an elevated level of apoptosis, which was measured as an increased number of RBL-2H3 cells immunopositive to activated caspase-3 after incubation with the cytokine mix over a period of 3 days compared to untreated controls (Fig. 5). Not only the epithelium is a target for mast cell mediators (Schultheiss et al., 2006), but also secretomotor neurons are equipped with receptors for mast cell mediators such as e.g. histamine (Bell et al., 2015). Indeed, anion secretion induced by antigen contact is strongly inhibited by the neurotoxin tetrodotoxin (Javed et al., 1992). So it might be thought that the impaired anion secretion in the presence of cytokines might be due to a reduced activation of secretomotor neurons. To elucidate mast cell - neuron interactions under inflammatory conditions, Ca2+ imaging experiments were performed and RBL-2H3 cells were cocultured with isolated submucosal ganglionic cells. In this coculture system, a mast cell degranulation evoked by compound 48/80 induces an increase in the cytosolic Ca2+ concentration of neighbouring neurons due to the release of mast cell mediators, mainly histamine and proteases (Bell et al., 2015). The neuronal response - measured as increase in the fura-2 ratio signal (Fig. 3) or as percentage of the neurons responding to mast cell degranulation (Fig. 4) - was significantly elevated, when the coculture was incubated with TNFα. Park et al. (2008) described a similar phenomenon: murine neurons, when incubated with TNFα, showed an enhanced Ca2+ signal after muscarinic stimulation due to upregulation of inositol 1,4,5-trisphosphate receptors. In contrast, when the coculture was incubated with the cytokine mix over a period of 1 d, neuronal activation after mast cell degranulation with compound 48/80 was significantly reduced (Fig. 3). These contrary effects might be due to changes in mast cell mediator release or changes in mast cell mediator composition, which indeed has been reported for patients with IBD (Bischoff et al., 1996a; Rijnierse et al., 2007) combined with changes in neuronal activity induced by proinflammatory cytokines.

IBD, but more recent studies showed that during IBD both active ion secretion and - to a larger extent - active ion absorption are impaired, resulting in a net fluid and electrolyte secretion (Martínez-Augustin et al., 2009). The mechanism underlying inhibited absorption is the downregulation of apical ion transporters during intestinal inflammation, such as Na+/H+ and Cl−/HCO3− exchanger or ENaCs, which are responsible for the uptake of Na+ and Cl− (Priyamvada et al., 2015; Anbazhagan et al., 2018). Interestingly, no differences could be observed at the expression on protein and mRNA level of chloride channels, such as CFTR (cystic fibrosis transmembrane conductance regulator), Ca2+-dependent Cl− channels, or the Na+-K+-2Clcotransporter, i.e. typical transporters involved in epithelial anion secretion (Sanchez de Medina et al., 2002; Hirota and McKay, 2009). Not only the basal Isc, but also the effects of different secretagogues on the intestinal epithelium, e.g. histamine, carbachol or forskolin, have been reported to be depressed during experimentally induced colitis or during IBD (Crowe et al., 1997; Sanchez de Medina et al., 2002; Pérez-Navarro et al., 2005; Hirota and McKay, 2009). In the present study, only IL-1β and the cytokine mix (after incubation for 1 or 2 h) diminished the response to carbachol (Fig. 1D). Carbachol, an agonist of muscarinic acetylcholine receptors, leads via an increase of the cytosolic Ca2+ concentration and the stimulation of Ca2+-activated K+ channels to an efflux of Cl− into the gut lumen (Hirota and McKay, 2006). The activity of these K+ channels, but also the activity of Na+K+-ATPase or protein kinase C, is reduced after induction of a colitis (Hirota and McKay, 2009). This might explain the diminished chloride secretion via the Ca2+ signaling pathway. In our study only the cytokine mix (applied for 2 h) was able to reduce the Isc evoked by the cAMP-dependent secretagogue forskolin (Fig. 1E). Forskolin, an activator of adenylate cyclase(s), stimulates Cl− secretion via protein kinase A-mediated phosphorylation of the apical CFTR channels so that Cl− can enter into the gut lumen (Hirota and McKay, 2006). The reduced response to forskolin might be due to the diminished activity of adenylate cyclase(s) or a reduced production of cAMP, which was found after induction of colitis (Sanchez de Medina et al., 2002). Interestingly, when the distal colon was incubated with the cytokine mix over a period of 3 h, there was no longer a reduction of both secretagogues, suggesting a compensatory mechanism of the intestinal epithelium (Fig. 1). 4.2. Effect of mast cells on epithelial and neuronal targets under inflammatory conditions in vitro Not only proinflammatory cytokines, but also mast cells (and their mediators) seem to be crucially involved in the pathogenesis of IBD. Thus, we investigated whether in vitro simulation of acute inflammation by cytokines might affect the secretory response of the epithelium evoked by mast cell degranulation or by histamine. Histamine, which is one of the main mast cell mediators, can induce a chloride secretion directly via histamine receptors on epithelial cells or indirectly via activation of submucosal neurons (Schultheiss et al., 2006; Bell et al., 2015). Although it has been observed that the histamine-induced Isc is increased in HT29 cells and in mouse colon in the presence of TNFα (Oprins et al., 2002), our findings showed an opposite effect: when rat distal colon was pretreated with IL-1β or the cytokine mix for 1 h, respectively, the histamine induced anion secretion was significantly reduced (Fig. 1C). This is, however, in accordance with other studies, where anion secretion evoked by histamine was impaired after induction of colitis with dextran sulphate sodium (DSS) in animal models or in specimens from patients with IBD (Crowe et al., 1997; Hirota and McKay, 2009). Consequently, cytokines seem to interfere with the histamine signaling pathway, e.g. by affecting the expression of histamine receptors or, what is more likely, the intracellular signaling pathway of histamine-induced chloride secretion, which is also mediated by Ca2+ (Schultheiss et al., 2006). Numerically (although without reaching statistical significance), the

4.3. Potential neuronal modulation of mast cells in the gut Different studies report that there is a bidirectional communication between mast cells and enteric neurons in the intestinal wall (Van Nassauw et al., 2007) and that these interactions might be involved in the pathophysiology of inflammatory bowel diseases (Rijnierse et al., 2007). Therefore, we asked the question whether the communication between enteric neurons and mast cells via neurotransmitters might be investigated in in vitro models. In a first step, the potential modulation of anion secretion induced by degranulation of mast cells physiologically present in different layers of the gut wall was investigated in Ussing chamber experiments. In these series of experiments preincubation of the tissue with nicotine (10−6 mol/l) strongly reduced the 11

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neurotransmitters, which modulate mast cell function. In future experiments, it might be interesting to investigate whether the communication between mast cells and epithelial cells might change simulating in vitro inflammatory conditions in coculture experiments, too.

increase in Isc evoked by the mast cell degranulator compound 48/80 (Fig. 6). This cannot be explained by an antisecretory effect of nicotine on the intestinal epithelium, as nicotine alone evoked an increase in Isc (Table 3), which is caused by stimulation of the Na+-K+-ATPase via epithelial nicotinic receptors (Lottig et al., 2019). A possible explanation for the reduced Isc evoked by compound 40/80 is the inhibitory effect of nicotinic acetylcholine receptors containing α7 subunits on the surface of mast cells, which are known to be negatively coupled to mast cell degranulation (Kageyama-Yahara et al., 2008). This was confirmed by experiments with RBL-2H3 cells in monoculture, where preincubation with nicotine strongly inhibited the increase in the cytosolic Ca2+ concentration (as initial event during mast cell degranulation) evoked by compound 48/80 (Fig. 8) and reduced the percentage of RBL-2H3 responding to this agent (Fig. 9). Physiologically, this response might contribute to the well-known anti-inflammatory effect of the vagus nerve and its transmitter acetylcholine during acute colitis via immune cell-dependent mechanisms (Ghia et al., 2006; Rahman et al., 2015). This is clinically obvious in the paradoxical, beneficial impact of smoking in patients, which suffer from ulcerative colitis (Berkowitz et al., 2018). In lung, where agonists of β2-adrenergic receptors play an essential role in the treatment of asthma bronchiale, catecholamines have been reported to inhibit mast cell degranulation (Hizawa, 2009). However, even in a high concentration (5·10−6 mol/l), where norepinephrine induces a stable negative Isc (Table 3) reflecting the well-known induction of K+ secretion by catecholamines (Hörger et al., 1998), norepinephrine did not affect the Isc induced by mast cell degranulation (Fig. 6), although about one third of isolated RBL-2H3 cells responded to the catecholamine with a transient increase in the cytosolic Ca2+ concentration (Table 4). Thus, modulation of intestinal mast cell functions by norepinephrine obviously is of no relevance in the coculture model used and probably also not in the gut wall under physiological conditions, as norepinephrine is only occasionally synthesized by enteric neurons (Furness, 2006). Muscarinic agonists (Bani-Sacchi et al., 1986) and substance P (Koon and Pothoulakis, 2006; Buhner and Schemann, 2012) have been found to evoke mast cell degranulation. Although both of them induced, when administered alone, an increase in the cytosolic Ca2+ concentration of cultured RBL-2H3 cells - which was especially strong in case of substance P (Table 4) - none of these agonists were able to enhance the secretory response evoked by compound 48/80 in Ussing chamber experiments. When considering the effect of both agonists on the increase of Ca2+ concentration (as initial step of mast cell degranulation) evoked by compound 48/80 at RBL-2H3 cells, at first glance a paradoxical response was observed, i.e. a reduction of the mean amplitude (Fig. 8) and - at least for substance P - a reduction of the percentage of the number of mast cells responding to the degranulating agent (Fig. 9). This paradoxical effect might, however, be well explained by the strong preactivation of the mast cells tested by prior administration of pilocarpine or substance P (Table 4), so that a large part of the mast cells is already preactivated by the respective agonist of neurotransmitter receptors. Nevertheless, the delayed response of RBL-2H3 cells located in direct neighbourhood of submucosal ganglia to neuronal stimulation via EFS clearly demonstrates the ability of the enteric nervous system to evoke mast cell activation also in the coculture system used here (Fig. 7).

Acknowledgement This study was supported by the Deutsche Forschungsgemeinschaft (DFG, grant Di 388/16–1). The diligent technical assistance of Miss B. Buss, B. Schmidt and A. Stockinger is a pleasure to acknowledge. References Ali, H., 2017. Emerging roles for MAS-related G protein-coupled receptor-X2 in host defense peptide, opioid, and neuropeptide-mediated inflammatory reactions. Adv. Immunol. 136, 123–162. https://doi.org/10.1016/bs.ai.2017.06.002. Anbazhagan, A.N., Priyamvada, S., Alrefai, W.A., Dudeja, P.K., 2018. Pathophysiology of IBD associated diarrhea. Tissue Barriers 6, e1463897. https://doi.org/10.1080/ 21688370.2018.1463897. Andres, H., Bock, R., Bridges, R.J., Rummel, W., Schreiner, J., 1985. Submucosal plexus and electrolyte transport across rat colonic mucosa. J. Physiol. 364, 301–312. https://doi.org/10.1113/jphysiol.1985.sp015746. Avula, L.R., Buckinx, R., Favoreel, H., Cox, E., Adriaensen, D., Van Nassauw, L., Timmermans, J.-P., 2013. Expression and distribution patterns of Mas-related gene receptor subtypes A-H in the mouse intestine: inflammation-induced changes. Histochem. Cell Biol. 139, 639–658. https://doi.org/10.1007/s00418-013-1086-9. Bani-Sacchi, T., Barattini, M., Bianchi, S., Blandina, P., Brunelleschi, S., Fantozzi, R., Mannaioni, P.F., Masini, E., 1986. The release of histamine by parasympathetic stimulation in Guinea-pig auricle and rat ileum. J. Physiol. 371, 29–43. https://doi.org/ 10.1113/jphysiol.1986.sp015960. Barmeyer, C., Amasheh, S., Tavalali, S., Mankertz, J., Zeitz, M., Fromm, M., Schulzke, J.D., 2004. IL-1beta and TNFalpha regulate sodium absorption in rat distal colon. Biochem. Biophys. Res. Commun. 317, 500–507. https://doi.org/10.1016/j.bbrc. 2004.03.072. Bauer, C., Duewell, P., Mayer, C., Lehr, H.A., Fitzgerald, K.A., Dauer, M., Tschopp, J., Endres, S., Latz, E., Schnurr, M., 2010. Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome. Gut 59, 1192–1199. https:// doi.org/10.1136/gut.2009.197822. Becker, J., Ott, D., Diener, M., 2019. Impact of sensitization and inflammation on the interaction of mast cells with the intestinal epithelium in rats. Front. Physiol. 10, 4969. https://doi.org/10.3389/fphys.2019.00329. Bell, A., Althaus, M., Diener, M., 2015. Communication between mast cells and rat submucosal neurons. Pflueg. Arch. Eur. J. Physiol. 467, 1809–1823. https://doi.org/10. 1007/s00424-014-1609-9. Berkowitz, L., Schultz, B.M., Salazar, G.A., Pardo-Roa, C., Sebastián, V.P., Álvarez-Lobos, M.M., Bueno, S.M., 2018. Impact of cigarette smoking on the gastrointestinal tract inflammation: opposing effects in Crohn's disease and ulcerative colitis. Front. Immunol. 9, 74. https://doi.org/10.3389/fimmu.2018.00074. Bernardazzi, C., Pego, B., De Souza, H.S.P., 2016. Neuroimmunomodulation in the gut: focus on inflammatory bowel disease. Mediat. Inflamm. 2016 1363818. https://doi. org/10.1155/2016/1363818. Bischoff, S.C., 2009. Physiological and pathophysiological functions of intestinal mast cells. Semin. Immunopathol. 31, 185–205. https://doi.org/10.1007/s00281-0090165-4. Bischoff, S.C., Schwengberg, S., Wordelmann, K., Weimann, A., Raab, R., Manns, M.P., 1996a. Effect of c-kit ligand, stem cell factor, on mediator release by human intestinal mast cells isolated from patients with inflammatory bowel disease and controls. Gut 38, 104–114. https://doi.org/10.1136/gut.38.1.104. Bischoff, S.C., Wedemeyer, J., Herrmann, A., Meier, P.N., Trautwein, C., Cetin, Y., Maschek, H., Stolte, M., Gebel, M., Manns, M.P., 1996b. Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease. Histopathology 28, 1–13. https://doi.org/10.1046/j.1365-2559.1996.262309.x. Blandina, P., Fantozzi, R., Mannaioni, P.F., Masini, E., 1980. Characteristics of histamine release evoked by acetylcholine in isolated rat mast cells. J. Physiol. 301, 281–293. https://doi.org/10.1113/jphysiol.1980.sp013205. Bode, H., Schmitz, H., Fromm, M., Scholz, P., Riecken, E., Schulzke, J., 1998. IL-1β and TNF-α, but not IFN-α, IFN-γ, IL-6 or IL-8, are secretory mediators in human distal colon. Cytokine 10, 457–465. https://doi.org/10.1006/cyto.1997.0307. Buhner, S., Schemann, M., 2012. Mast cell–nerve axis with a focus on the human gut. Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1822, 85–92. https://doi.org/10. 1016/j.bbadis.2011.06.004. Butchers, P.R., Vardey, C.J., Johnson, M., 1991. Salmeterol: a potent and long-acting inhibitor of inflammatory mediator release from human lung. Br. J. Pharmacol. 104, 672–676. https://doi.org/10.1111/j.1476-5381.1991.tb12487.x. Crowe, S.E., Luthra, G.K., Perdue, M.H., 1997. Mast cell mediated ion transport in intestine from patients with and without inflammatory bowel disease. Gut 41, 785–792. https://doi.org/10.1136/gut.41.6.785. Da Silva, E.Z.M., Jamur, M.C., Oliver, C., 2014. Mast cell function: a new vision of an old cell. J. Histochem. Cytochem. 62, 698–738. https://doi.org/10.1369/ 0022155414545334. De Winter, B.Y., Van den Wijngaard, R.M., De Jonge, W.J., 2012. Intestinal mast cells in gut inflammation and motility disturbances. Biochim. Biophys. Acta (BBA) - Mol.

4.4. Outlook Overall, the present experiments reveal that the interactions between mast cells and submucosal neurons, as one of their main targets during mast cell mediator-induced epithelial anion secretion, are modified in the presence of proinflammatory cytokines. Apoptosis of mast cells and changes in the responsiveness of submucosal neurons to mast cell mediators are involved in this process. Moreover, there is a communication from submucosal neurons to mast cells via 12

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Oprins, J.C.J., Van der Burg, C., Meijer, H.P., Munnik, T., Groot, J.A., 2002. Tumour necrosis factor α potentiates ion secretion induced by histamine in a human intestinal epithelial cell line and in mouse colon: involvement of the phospholipase D pathway. Gut 50, 314–321. https://doi.org/10.1136/gut.50.3.314. Park, K.M., Yule, D.I., Bowers, W.J., 2008. Tumor necrosis factor-alpha potentiates intraneuronal Ca2+ signaling via regulation of the inositol 1,4,5-trisphosphate receptor. J. Biol. Chem. 283, 33069–33079. https://doi.org/10.1074/jbc. M802209200. Pérez-Navarro, R., Ballester, I., Zarzuelo, A., Sánchez de Medina, F., 2005. Disturbances in epithelial ionic secretion in different experimental models of colitis. Life Sci. 76, 1489–1501. https://doi.org/10.1016/j.lfs.2004.09.019. Priyamvada, S., Gomes, R., Gill, R.K., Saksena, S., Alrefai, W.A., Dudeja, P.K., 2015. Mechanisms underlying dysregulation of electrolyte absorption in inflammatory bowel disease-associated diarrhea. Inflamm. Bowel Dis. 21, 2926–2935. https://doi. org/10.1097/MIB.0000000000000504. Rahman, A.A., Robinson, A.M., Jovanovska, V., Eri, R., Nurgali, K., 2015. Alterations in the distal colon innervation in Winnie mouse model of spontaneous chronic colitis. Cell Tissue Res. 362, 497–512. https://doi.org/10.1007/s00441-015-2251-3. Rijnierse, A., Nijkamp, F.P., Kraneveld, A.D., 2007. Mast cells and nerves tickle in the tummy. Pharmacol. Ther. 116, 207–235. https://doi.org/10.1016/j.pharmthera. 2007.06.008. Salim, S.Y., Söderholm, J.D., 2011. Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflamm. Bowel Dis. 17, 362–381. https://doi.org/10. 1002/ibd.21403. Sanchez de Medina, F., Perez, R., Martinez-Augustin, O., Gonzalez, R., Lorente, M.D., Galvez, J., Zarzuelo, A., 2002. Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP. Pflüg. Arch. 444, 378–388. https://doi. org/10.1007/s00424-002-0807-z. Schemann, M., Michel, K., Ceregrzyn, M., Zeller, F., Seidl, S., Bischoff, S.C., 2005. Human mast cell mediator cocktail excites neurons in human and Guinea-pig enteric nervous system. Neuro Gastroenterol. Motil. 17, 281–289. https://doi.org/10.1111/j.13652982.2004.00591.x. Schmitz, H., Fromm, M., Bentzel, C.J., Scholz, P., Detjen, K., Mankertz, J., Bode, H., Epple, H.J., Riecken, E.O., Schulzke, J.D., 1999. Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J. Cell Sci. 112, 137–146. Schmitz, H., Fromm, M., Bode, H., Scholz, P., Riecken, E.O., Schulzke, J.D., 1996. Tumor necrosis factor-α induces Cl- and K+ secretion in human distal colon driven by prostaglandin E2. Am. J. Physiol. 271, G669–G674. https://doi.org/10.1152/ajpgi. 1996.271.4.G669. Senyshyn, J., Baumgartner, R.A., Beaven, M.A., 1998. Quercetin sensitizes RBL-2H3 cells to polybasic mast cell secretagogues through increased expression of Gi GTP-binding proteins linked to a phospholipase C signaling pathway. J. Immunol. 160, 5136–5144. Swieter, M., Midura, R.J., Nishikata, H., Oiver, C., Berenstein, E.H., Mergenhagen, S.E., Hascall, V.C., Siraganian, R.P., 1993. Mouse 3T3 fibroblasts induce rat basophilic leukemia (RBL-2H3) cells to acquire responsiveness to compound 48/80. J. Immunol. 150, 617–624. Schultheiss, G., Diener, M., 2000. Adrenoceptor-mediated secretion across the rat colonic epithelium. Eur. J. Pharmacol. 403, 251–258. https://doi.org/10.1016/s00142999(00)00487-8. Schultheiss, G., Hennig, B., Schunack, W., Prinz, G., Diener, M., 2006. Histamine-induced ion secretion across rat distal colon: involvement of histamine H1 and H2 receptors. Eur. J. Pharmacol. 546, 161–170. https://doi.org/10.1016/j.ejphar.2006.07.047. Singh, U.P., Singh, N.P., Murphy, E.A., Price, R.L., Fayad, R., Nagarkatti, M., Nagarkatti, P.S., 2016. Chemokine and cytokine levels in inflammatory bowel disease patients. Cytokine 77, 44–49. https://doi.org/10.1016/j.cyto.2015.10.008. Van Nassauw, L., Adriaensen, D., Timmermans, J.-P., 2007. The bidirectional communication between neurons and mast cells within the gastrointestinal tract. Auton. Neurosci. 133, 91–103. https://doi.org/10.1016/j.autneu.2006.10.003. Wittkopf, N., Neurath, M.F., Becker, C., 2014. Immune-epithelial crosstalk at the intestinal surface. J. Gastroenterol. 49, 375–387. https://doi.org/10.1007/s00535-0130929-4. Yamamoto, T., Kodama, T., Lee, J., Utsunomiya, N., Hayashi, S., Sakamoto, H., Kuramoto, H., Kadowaki, M., 2014. Anti-allergic role of cholinergic neuronal pathway via α7 nicotinic ACh receptors on mucosal mast cells in a murine food allergy model. PLoS One 9, e85888. https://doi.org/10.1371/journal.pone.0085888.

Basis Dis. 1822, 66–73. https://doi.org/10.1016/j.bbadis.2011.03.016. Fowler, C.J., Sandberg, M., Tiger, G., 2003. Effects of water-soluble cigarette smoke extracts upon the release of ß-hexosaminidase from RBL-2H3 basophilic leukaemia cells in response to substance P, compound 48/80, concanavalin A and antigen stimulation. Inflamm. Res. 52, 461–469. https://link.springer.com/article/10.1007% 2Fs00011-003-1202-8. Furness, J.B., 2006. The Enteric Nervous System. Blackwell, Oxford. Ghia, J.E., Blennerhassett, P., Kumar–Ondiveeran, H., Verdu, E.F., Collins, S.M., 2006. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology 131, 1122–1130. https://doi.org/10. 1053/j.gastro.2006.08.016. Gupta, S., 2002. A decision between life and death during TNF-alpha-induced signaling. J. Clin. Immunol. 22, 185–194. https://doi.org/10.1023/A:1016089607548. He, S.-H., 2004. Key role of mast cells and their major secretory products in inflammatory bowel disease. World J. Gastroenterol. 10, 309. https://doi.org/10.3748/wjg.v10.i3. 309. Hirota, C.L., McKay, D.M., 2006. Cholinergic regulation of epithelial ion transport in the mammalian intestine. Br. J. Pharmacol. 149, 463–479. https://doi.org/10.1038/sj. bjp.0706889. Hirota, C.L., McKay, D.M., 2009. Loss of Ca-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca-activated K channel opener. Br. J. Pharmacol. 156, 1085–1097. https://doi.org/10.1111/j.1476-5381.2009.00122.x. Hizawa, N., 2009. Beta-2 adrenergic receptor genetic polymorphisms and asthma. J. Clin. Pharm. Ther. 34, 631–643. https://doi.org/10.1111/j.1365-2710.2009.01066.x. Hörger, S., Schultheiss, G., Diener, M., 1998. Segment-specific effects of epinephrine on ion transport in the colon of the rat. Am. J. Physiol. Gastrointest. Liver Physiol. 275, G1367–G1376. https://doi.org/10.1152/ajpgi.1998.275.6.G1367. Hug, F., Diener, M., Scharrer, E., 1996. Modulation by fish oil diet of eicosanoid-induced anion secretion in the rat distal colon. Z. Ernahrungswiss. 35, 323–331. https://doi. org/10.1007/BF01610550. Javed, N.H., Wang, Y.Z., Cooke, H.J., 1992. Neuroimmune interactions: role for cholinergic neurons in intestinal anaphylaxis. Am. J. Physiol. 263, G847–G852. https:// doi.org/10.1152/ajpgi.1992.263.6.G847. Kageyama-Yahara, N., Suehiro, Y., Yamamoto, T., Kadowaki, M., 2008. IgE-induced degranulation of mucosal mast cells is negatively regulated via nicotinic acetylcholine receptors. Biochem. Biophys. Res. Commun. 377, 321–325. https://doi.org/10.1016/ j.bbrc.2008.10.004. König, J., Wells, J., Cani, P.D., Garcia-Rodenas, C.L., MacDonald, T., Mercenier, A., Whyte, J., Troost, F., Brummer, R.-J., 2016. Human intestinal barrier function in health and disease. Clin. Transl. Gastroenterol. 7, e196. https://doi.org/10.1038/ctg. 2016.54. Koon, H.W., Pothoulakis, C., 2006. Immunomodulatory properties of substance P: the gastrointestinal system as a model. Ann. N. Y. Acad. Sci. 1088, 23–40. https://doi. org/10.1196/annals.1366.024. Kurashima, Y., Kiyono, H., 2014. New era for mucosal mast cells: their roles in inflammation, allergic immune responses and adjuvant development. Exp. Mol. Med. 46, e83. https://doi.org/10.1038/emm.2014.7. Lottig, L., Bader, S., Jimenez, M., Diener, M., 2019. Evidence for metabotropic function of epithelial nicotinic cholinergic receptors in rat colon. Br. J. Pharmacol. 176, 1328–1340. https://doi.org/10.1111/bph.14638. Martínez-Augustin, O., Romero-Calvo, I., Suárez, M.D., Zarzuelo, A., De Medina, F.S., 2009. Molecular bases of impaired water and ion movements in inflammatory bowel diseases. Inflamm. Bowel Dis. 15, 114–127. https://doi.org/10.1002/ibd.20579. McNeil, B.J., Pundir, P., Meeker, S., Han, L., Undem, B.J., Kulka, M., Dong, X., 2015. Identification of a mast cell specific receptor crucial for pseudo-allergic drug reactions. Nature 519, 237–241. Mishra, N.C., Rir-sima-ah, J., Boyd, R.T., Singh, S.P., Gundavarapu, S., Langley, R.J., Razani-Boroujerdi, S., Sopori, M.L., 2010. Nicotine inhibits Fcε RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through α7/α 9/α10-nicotinic receptors. J. Immunol. 185, 588–596. https://doi.org/ 10.4049/jimmunol.0902227. Neurath, M.F., Fuss, I., Pasparakis, M., Alexopoulou, L., Haralambous, S., Meyer zum Büschenfelde, K.H., Strober, W., Kollias, G., 1997. Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice. Eur. J. Immunol. 27, 1743–1750. https://doi.org/10.1002/eji.1830270722. Oda, S., Uchida, K., Wang, X., Lee, J., Shimada, Y., Tominaga, M., Kadowaki, M., 2013. TRPM2 contributes to antigen-stimulated Ca2⁺ influx in mucosal mast cells. Pflüg. Arch. 465, 1023–1030. https://doi.org/10.1007/s00424-013-1219-y.

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