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Neurogastroenterology
Original article
Histamine H4 and H1 receptors contribute to postinflammatory visceral hypersensitivity
  1. Annemie Deiteren1,
  2. Joris G De Man1,
  3. Nathalie E Ruyssers1,
  4. Tom G Moreels1,2,
  5. Paul A Pelckmans1,2,
  6. Benedicte Y De Winter1
  1. 1Division of Gastroenterology, Laboratory of Experimental Medicine and Pediatrics, University of Antwerp, Antwerp, Belgium
  2. 2Department of Gastroenterology, Antwerp University Hospital, Antwerp, Belgium
  1. Correspondence to Professor Benedicte De Winter, Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, Antwerp B-2610, Belgium; benedicte.dewinter{at}uantwerpen.be

Abstract

Objectives Substantial evidence implicates mast cells and their main constituent histamine in the pathogenesis of visceral hypersensitivity. We explored the specific contribution of histamine H4 (H4R) and H1 (H1R) receptors to visceral hypersensitivity in a postinflammatory rat model.

Design Trinitrobenzenesulfonic acid (TNBS)-colitis was monitored individually by colonoscopy: first on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Experiments were performed 3 days after endoscopic resolution of colitis. Visceral sensitivity was assessed by quantifying visceromotor responses (VMRs) to colorectal distension. Colonic mast cell numbers, histamine release and H4R and H1R mRNA expression were quantified. JNJ7777120 (H4R antagonist) and/or levocetirizine (H1R antagonist) were administered 30 min prior to VMR assessment or histamine release assay.

Results Postcolitis rats displayed a higher number of colonic mast cells, excessive histamine release and significantly enhanced VMRs. Heightened VMRs were dose-dependently reduced by JNJ7777120 and levocetirizine; combined administration of JNJ7777120 and levocetirizine potentiated the antinociceptive effect. In the colon, both H4R and H1R mRNA were present; in the dorsal root ganglia, only H1R mRNA was found. Only colonic H4R mRNA expression was increased in postcolitis rats. Excessive histamine release in postcolitis rats was attenuated by the highest dose of JNJ7777120.

Conclusions H4R and H1R antagonists dose-dependently reduce and even normalise postinflammatory visceral hypersensitivity via different underlying mechanisms but with a synergistic effect. Both receptor subtypes represent promising targets for the treatment of postinflammatory visceral hypersensitivity.

  • VISCERAL HYPERSENSITIVITY
  • MAST CELLS
  • HISTAMINE
  • VISCERAL NOCICEPTION

https://doi.org/10.1136/gutjnl-2013-305870

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    Significance of this study

    What is already known on this subject?

    • Mast cells and their main mediator histamine contribute significantly to visceral hypersensitivity.

    • A recent clinical trial highlights the therapeutic potential of H1 receptor (H1R) antagonists for the treatment of IBS symptoms.

    What are the new findings?

    • Selective blockade of the H4R or H1R dose-dependently reduced in vivo postinflammatory visceral hypersensitivity in a rat model, and there was a functional interplay between both receptor subtypes.

    • Only H1R mRNA was present in the dorsal root ganglia, whereas both H4R and H1R mNRA were present in the colon. Colonic H4R mRNA expression was increased in the postinflammation group.

    How might it impact on clinical practice in the foreseeable future?

    • Our findings reveal a therapeutic potential of selective H4R and H1R-targeted therapies, alone or in combination, for postinflammatory visceral hypersensitivity, supporting the rationale for H1R-directed clinical trials and uncovering a novel target, the H4R.

    Introduction

    IBS is a functional gastrointestinal disorder characterised by chronic abdominal pain and an altered bowel habit. Although the exact pathophysiology is yet to be elucidated, visceral hypersensitivity, disturbed intestinal motor function and psychological aspects have been identified as important contributors.1 Visceral hypersensitivity is considered the main factor underlying abdominal pain in IBS and, indeed, enhanced pain perception in response to colorectal distension is present in the majority of IBS patients and has been shown to correlate with abdominal pain symptoms.1–3 However, treatment options for IBS-related visceral pain are still limited and patients and physicians eagerly await novel therapeutic strategies.4

    A potential target in this quest for new therapies is the mast cell (MC) and its main mediator histamine. Several lines of evidence corroborate MC involvement in visceral hypersensitivity.5–10 Both the location of MCs in close apposition to afferent nerves in the gut wall and the bidirectional communication between them both point towards a potential role for MCs in the sensitisation of afferent nerves.5 ,6 Moreover, activated MCs in close proximity to the colonic nerves were shown to correlate with the severity and frequency of abdominal pain in IBS patients.7 Functionally, mucosal MCs obtained from IBS patients show a higher rate of activation and release greater amounts of histamine and tryptase.7 In addition, IBS colonic biopsy supernatants, containing increased levels of these MC mediators, excite dorsal root ganglia (DRG) neurons, increase the firing rate of mesenteric afferent nerves and induce visceral hyperalgesia when applied in animal models.8–10 Moreover, the excitatory action of IBS supernatant on guinea pig enteric neurons was more pronounced for hypersensitive compared with normosensitive patients and correlated with patients’ degree of visceral hypersensitivity.11

    Histamine is the main MC mediator and binds to four receptor subtypes (H1-4) of which H1 and H4 may be interesting targets for visceral pain. Recently, ketotifen, a MC stabiliser with H1 receptor (H1R) antagonistic properties, increased the threshold of discomfort to rectal distension in IBS patients with documented visceral hypersensitivity and improved IBS symptoms such as abdominal pain.12 Interestingly, ketotifen did not affect MC numbers or their mediator release, leading the authors to suggest that the beneficial effect of ketotifen was mediated by its H1R antagonistic properties rather than by stabilising mucosal MCs.13 This could well be the case, as preliminary results from a 12-week clinical trial indicate that ebastin, a selective H1R antagonist, improves abdominal pain in IBS patients.14

    H4 receptors (H4R) are currently under investigation for the treatment of immune-mediated disorders as pruritus, asthma, allergic rhinitis and rheumatoid arthritis.15 ,16 Present on immune cells, among which MCs, on endocrine cells and on neurons, H4Rs are expressed throughout the gastrointestinal tract.17 In addition, pharmacological in vitro studies indicate that they are functionally active in the human submucosal plexus.18 Moreover, preliminary in vivo data demonstrate that in the GI tract, H4Rs mediate indomethacin-induced gastric mucosal damage, modulate ischaemia/reperfusion-induced intestinal damage and zymosan-induced peritonitis and participate in gut inflammation in acute trinitrobenzenesulfonic acid (TNBS)-colitis.19–21 While their role in inflammation has been extensively studied, H4R involvement in visceral hypersensitivity has not been investigated to date. In this study, we therefore aimed to explore the specific contributions of H4Rs and H1Rs to visceral pain perception in a rat model of postinflammatory visceral hypersensitivity.

    Methods

    Animals

    Male Sprague–Dawley rats (200–225 g, Charles River, France) were allowed to acclimatise to housing conditions for 1 week prior to experimentation. Animals had free access to water and food and were kept at constant room temperature (22±2°C) and humidity (60%) and on a 12:12 hour light/dark cycle. All experiments were approved by the Committee for Medical Ethics and the use of Experimental Animals at the University of Antwerp (file number 2010-18).

    Colitis

    Distal colitis was induced by intrarectal instillation of 0.5 mL of 15 mg TNBS in 50% ethanol under pentobarbital anaesthesia (60 mg/kg intraperitoneally). Control animals received a 0.5 mL saline enema.

    Colonoscopy

    After pentobarbital sedation (45 mg/kg intraperitoneally), colonoscopy was performed using a baby gastroscope (Olympus Europa GmbH, Germany). After lubrication and under direct colonoscopic vision, the endoscope was gently introduced into the distal 10 cm of the colon. During withdrawal, mucosal damage was assessed using our previously published scoring system (score 0–19).22 ,23

    Postmortem evaluation of inflammatory markers

    At the end of the experiment, macroscopic mucosal damage was assessed using a standardised scoring system (score 0–10).22 A representative segment was fixed in 4% formaldehyde, embedded in paraffin for H&E staining (5 µm) and scored microscopically for the presence of an inflammatory infiltrate, the number of layers infiltrated, mucosal architectural distortion and oedema (score 0–10).22 The postinflammatory status was additionally confirmed in paraffin-embedded sections by immunohistochemical detection of CD3+ cells using anti-CD3 (Abcam; 1:300 dilution) as the primary antibody and a biotin-conjugated antirabbit polyclonal secondary antibody. A colour image analysis system was used to quantify the CD3-positive area (ImageJ 1.47v, USA).

    Myeloperoxidase (MPO) activity, which is directly related to the number and activity of myeloid cell infiltration, was assayed in colonic specimens as previously described in detail and expressed as units per gram tissue (U/g tissue).23 ,24 Finally, colonic levels of IL-2 and IL-17a were quantified by ELISA (eBioscience, Austria) and expressed as pg/g tissue as these cytokines have previously been shown to be upregulated during acute TNBS-colitis.25–27

    MCs and histamine release

    A 1 cm colon segment was fixed in Carnoy's solution and embedded in paraffin. Transverse sections (5 µm) were stained with toluidine blue (pH 0.5) for identification of MCs.

    The number of MCs was evaluated in 10 non-overlapping fields (0.136 mm2/field) at ×400 magnification by an observer blinded to the study groups and expressed per mm².

    For in vitro quantification of the spontaneous histamine release, a segment of distal colon (1 cm) was incubated in Krebs–Ringer solution for 1 h at 37°C. Histamine levels in the supernatant were evaluated by ELISA (Immunotech, France) and expressed as nM/mg tissue.

    Visceral sensitivity

    The visceromotor response (VMR) to colorectal balloon distension was used as an objective measure of visceral sensitivity in fully awake animals.23 ,28 Three days prior to VMR assessment, two EMG electrodes (Advent Research Materials Ltd, UK) were sutured into the external oblique abdominal muscle under deep pentobarbital anaesthesia (60 mg/kg) and exteriorised dorsally. On the day of VMR assessment, rats were mildly restrained using a fabric glove. A lubricated, latex balloon (length 5 cm) was introduced in the distal colon, up to 0.5 cm passed the anal verge and connected to a barostat system (Distender Series II Barostat, G&J Electronics, Canada) for balloon distension (10–80 mm Hg, 20 s, 4 min interval). The electrodes were relayed to a data acquisition system and the corresponding EMG signal was recorded, amplified (Neurolog, Digitimer Ltd, UK) and digitised (CED 1401, Cambridge Electronic Design, UK) to a PC for off-line analysis using Spike2 V.5.16 (Cambridge Electronic Design, UK). After correction for movement and breathing, the analogue EMG signal was rectified and integrated. To quantify the magnitude of the VMR at each distension pressure, the area under the curve (AUC) immediately before the distension (20 s) was subtracted from the AUC during the balloon distension (20 s).

    Colonic compliance

    Colonic compliance was studied in the same animal to exclude pharmacologically mediated changes in the viscoelastic properties of the colonic wall as a potential antinociceptive mechanism. Rats were anesthetised (pentobarbital 45 mg/kg) and graded volumes (0–2.5 mL) were applied to the balloon inserted in the colorectum while recording the corresponding intracolonic pressure.23 ,29

    Quantitative RT-PCR

    The mRNA expression of H1R and H4R was quantified in the colon and DRGs from controls and postcolitis rats. The DRGs contain the primary afferent neurons conveying sensory information from the colon to the spinal cord and were harvested bilaterally at Th13-L2 (splanchnic afferents) and L6-S1 (pelvic afferents). Total RNA was extracted from colon using the RNeasy Minikit (Qiagen, The Netherlands) and from DRGs using the Absolutely RNA microprep kit (Stratagene, USA). RNA was then converted to cDNA (Transcriptor First Strand cDNA Synthese Kit; Roche, Belgium). A Taqman gene expression assay was performed for H1R (Rn00566691_s1; Applied Biosystems, USA) and H4R (Rn00590929_m1; Applied Biosystems, USA) on a ABIPrism 7300 sequent detector system (Applied Biosystems) in a 25 µL reaction volume containing 2 µL cDNA, 12.5 µL TaqMan Universal PCR master mix (Applied Biosystems), 1.25 µL Taqman assay probe and 9.25 µL RNase-free H2O. The parameters for PCR amplification were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

    Expression of H1R and H4R was normalised against the housekeeping gene β-actin (Rn00667869_m1; Applied Biosystems) for calculation of comparative cycle thresholds (ΔCT=CT(H1R or H4R)—CT(β-actin)). Relative expression of mRNA species was then determined as 2−ΔΔCT with ΔΔCT=ΔCT (postcolitis)−ΔCT (control).30

    Experimental design

    A scheme of the experimental design is presented in figure 1. Rats were randomised to receive a saline (control) or TNBS instillation. The extent of colitis was colonoscopically verified on day 3. From day 10 onwards, convalescence was monitored individually by repeated colonoscopy that was performed every 4 days to determine for each individual animal whether endoscopic colonic healing had occurred. When mucosal healing was present on colonoscopic examination, further experiments were performed 3 days later. Three series of experiments were conducted in three distinct sets of rats (figure 1).

    Figure 1
    Figure 1

    Schematic of the experimental design. After the induction of trinitrobenzenesulfonic acid (TNBS) (colitis) or saline (control), animals were monitored individually by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence until full resolution of mucosal damage had occurred. Three days after resolution of colitis, further experiments were conducted. In total, three experimental series were performed. Rats assigned to the first set did not receive drug or vehicle, while animals in the second and third experimental groups were administered drug or vehicle 30 min prior to the start of the visceromotor response (VMR) distension protocol (set 2) or 30 min prior to sacrifice (set 3).

    In the first set of rats, colonic tissue and DRGs were harvested 3 days after endoscopic healing to quantify MC numbers, spontaneous histamine release and H4R and H1R mRNA expression. Also, macroscopic and microscopic evaluation of inflammation was performed to confirm the postinflammatory status in addition to an MPO activity assay, CD3-staining and quantification of IL-2 and IL-17a, which have previously been shown to be increased during acute TNBS-colitis.25 ,31 N=10 in each group.

    The second set of rats was injected intraperitoneally 3 days after endoscopic healing (i) with JNJ7777120 (10–140 mg/kg; T1/2 ≈2 h, bioavailability 22%), a selective H4R antagonist or its vehicle (30% 2-hydroxypropyl-β-cyclodextrin); (ii) with levocetirizine (0.01–1 mg/kg; T1/2 ≈8 h, bioavailability >90%), a selective H1R antagonist or its vehicle (saline); or (iii) with a combination of both antagonists (JNJ7777120 10 mg/kg plus levocetirizine 0.1 mg/kg) or vehicle (30% 2-hydroxypropyl-β-cyclodextrin). VMRs were assessed 30 min later. Colonic compliance was evaluated immediately following the VMR distension protocol in rats that had received the highest drug dose or its vehicle. At the end of the experiments, rats were sacrificed to harvest colonic tissue for macroscopic and microscopic analysis. N=4–9 in each group.

    In a third set of rats, JNJ7777120 or levocetirizine, or their respective vehicles, were administered 3 days after endoscopic healing, and rats were sacrificed 30 min later. Spontaneous colonic histamine release was assessed in vitro in addition to the macroscopic and microscopic evaluation of the colon. N=8–10 in each group.

    Solutions and drugs

    JNJ7777120 was kindly gifted by Janssen Research & Development. Levocetirizine was purchased from UCB Pharma, Belgium. TNBS was bought from Fluka, Germany. Pentobarbital was obtained from Ceva, Belgium. 2-Hydroxypropyl-β-cyclodextrin, hexadecyltrimethyl-ammonium bromide and o-dianisidine dihydrochloride were purchased from Sigma-Aldrich Inc, USA, and hydrogen peroxide from Merck, Germany.

    Statistical analysis

    Data are presented as mean±SEM. Variables were analysed using unpaired Student t test and two-way ANOVA, followed by Student–Newman–Keuls (SNK) posthoc test when appropriate. Analysis of VMR and compliance data was performed by the generalised estimating equation model, followed by least significant difference (LSD) posthoc test when appropriate. Statistical analysis was executed using SPSS V.18.0 software. Statistical significance was set at p<0.05.

    Results

    MC numbers and histamine release are increased after the resolution of TNBS-colitis

    In the first set of experiments, a mild colitis characterised by multiple serpiginous ulcers was present 3 days after TNBS-instillation and resolved spontaneously after a mean period of 11 days (range 10–22). Analysis of inflammation markers (colonoscopy, macroscopy and microscopy, MPO activity, CD3 expression and IL-2 and IL-17a levels) confirmed the postinflammatory status at the time of the experiment (table 1).

    View this table:
    Table 1

    Confirmation of the postinflammatory status

    MCs were present in the colon of both control and postinflammatory rats with the following distribution: mucosa>submucosa>muscularis externa (figure 2A,B). The number of MCs was significantly increased in the distal colon of postcolitis rats compared with controls. In addition, the spontaneous release of histamine was threefold higher in specimens obtained from postcolitis rats compared with controls (figure 3A).

    Figure 2
    Figure 2

    (A) Toluidine blue staining demonstrating the presence of mast cells (*) in the colonic mucosa of a control and a postcolitis rat. (B) The number of mast cells (MC) in the mucosa, submucosa and muscularis externa in controls (open bars) and after the resolution of trinitrobenzenesulfonic acid (TNBS)-colitis (hatched bars). Two-way ANOVA followed by Student–Newman–Keuls posthoc test; n=6–7; ###p<0.001, significant effect of the factor colitis; *p<0.05, significant effect of the factor layer, posthoc comparison; no significant interaction between layer and colitis.

    Figure 3
    Figure 3

    (A) Spontaneous release of histamine from whole-mount distal colon in controls and after the resolution of trinitrobenzenesulfonic acid (TNBS)-colitis. Unpaired Student t test; n=10; **p<0.01, significantly different from controls. (B) The effect of JNJ7777120 (35 and 140 mg/kg) and its vehicle on spontaneous in vitro histamine release in the distal colon. Two-way ANOVA followed by Student–Newman–Keuls posthoc test; n=9–10; significant interaction between colitis and drug; **p<0.01, significantly different from control+vehicle. (C) The effect of levocetirizine (0.1 and 1 mg/kg) and its vehicle on histamine release in vitro. Two-way ANOVA followed by SNK posthoc test; n=8–10; **p<0.01, significant effect of the factor colitis; no significant effect of the factor drug; no significant interaction.

    H4Rs contribute to postinflammatory visceral hypersensitivity

    After resolution of the TNBS-induced colitis (mean time to mucosal healing 15 days; range 10–26 days), which was confirmed by colonoscopy and postmortem inflammatory markers (table 2), VMRs were significantly increased in vehicle-treated rats that had recovered from colitis compared with controls for all distension pressures (10–80 mm Hg), indicating the presence of postinflammatory visceral hypersensitivity (figure 4A). This visceral hypersensitivity was dose-dependently reduced by JNJ7777120 in postcolitis rats: 10 mg/kg had no significant effect on VMRs, whereas 35 mg/kg effectively reversed increased VMRs at the lower distension pressures (10 and 20 mm Hg) and 70 and 140 mg/kg normalised VMRs over the full range of distension pressures (10–80 mm Hg) (figure 4A–D). In contrast, in controls JNJ7777120 did not affect VMRs significantly (figure 4E). Colonic compliance was similar in control and postcolitis rats and was not modified by 140 mg/kg of JNJ7777120 (figure 4F). In addition, JNJ7777120 treatment did not affect the tissue's colonoscopic, macroscopic and microscopic appearance at any doses tested (table 2). In the distal colon of postcolitis rats, H4R mRNA expression levels were increased compared with controls (table 3); however, no H4R mRNA could be demonstrated in the DRGs of either group. The increased spontaneous release of histamine in the distal colon of rats that had recovered from colitis remained equally elevated after 35 mg/kg of JNJ7777120, whereas it was no longer significantly different from controls after 140 mg/kg of JNJ7777120 (figure 3B).

    View this table:
    Table 2

    Postinflammatory status for animals receiving JNJ7777120 or vehicle

    View this table:
    Table 3

    mRNA expression of H4R and H1R in colon and dorsal root ganglias (Th13-L2 and L6-S1)

    Figure 4
    Figure 4

    The effect of JNJ7777120 (10–140 mg/kg) on visceromotor responses (VMRs) in postcolitis (A–D) and control rats (E). To facilitate comparison, VMRs for vehicle-treated controls are also shown in A–D (gray dashed line). Generalised estimating equations, least significant difference posthoc test, n=8–9; #p<0.05, ##p<0.01, ###p<0.001, significantly different from control+vehicle. *p<0.05, **p<0.01, ***p<0.001, significantly different from postcolitis+vehicle. (F) The effect of 140 mg/kg JNJ7777120 and its vehicle on colonic compliance in control and postcolitis groups; no significant effect of the factor drug or colitis.

    H1Rs contribute to postinflammatory visceral hypersensitivity

    Again, significant visceral hypersensitivity was present in rats that had recovered from colitis (mean time to mucosal healing 13 days, range 10–26 days) and were only vehicle-treated (figure 5A). This postinflammatory hypersensitivity was not altered by the lowest dose of levocetirizine (0.01 mg/kg), while 0.1 and 1 mg/kg normalised heightened VMRs (figure 5A–C). The highest dose of 1 mg/kg did not affect visceral sensitivity in controls, nor did it modify colonic compliance (figure 5D,E). Levocetirizine at the different doses tested did not influence postmortem inflammatory parameters, which were in keeping with the postinflammatory status (table 4).

    View this table:
    Table 4

    Postinflammatory status for animals receiving levocetirizine or vehicle

    Figure 5
    Figure 5

    The effect of levocetirizine (0.01–1 mg/kg) on visceromotor responses (VMRs) in postcolitis (A–C) and control rats (D). To facilitate comparison, VMRs for vehicle-treated controls are also shown in A–C (gray dashed line). generalised estimating equations, least significant difference posthoc test, n=7–9; #<p<0.05, ##p<0.01, ###p<0.001, significantly different from control+vehicle. *p<0.05, **p<0.01, ***p<0.001, significantly different from postcolitis+vehicle. (E) The effect of 1 mg/kg levocetirizine and its vehicle on colonic compliance in control and postcolitis groups; no significant effect of the factor drug or colitis.

    The relative expression of H1R mRNA was comparable in the colon and DRGs of control and postcolitis rats at Th13-L2 and L6-S1 (table 3). Histamine release in the distal colon was significantly increased in the postcolitis group and was not significantly attenuated by levocetirizine, although histamine levels tended to be decreased after the 1 mg/kg dose (figure 3C).

    Combined H4/H1R antagonism potentiates the antinociceptive effect

    Significant visceral hypersensitivity, which was present after the resolution of TNBS-colitis (mean time to mucosal healing 13 days, range 10–14 days), was significantly reduced by the combined administration of 10 mg/kg of JNJ7777120 and 0.01 mg/kg of levocetirizine (figure 6A). In contrast, this treatment did not modify VMRs in control, nor altered colonic compliance (figure 6B,C). Postinflammatory markers were not affected by the combined administration (table 5).

    View this table:
    Table 5

    Postinflammatory status for animals receiving combined treatment with JNJ7777120 (10 mg/kg) and levocetirizine (0.01 mg/kg) or vehicle

    Figure 6
    Figure 6

    The effect of combined administration of JNJ7777120 (10 mg/kg) and levocetirizine (0.01 mg/kg) on visceromotor responses (VMRs) in postcolitis (A) and control rats (B). To facilitate comparison, VMRs for vehicle-treated controls are also shown in (A) (gray dashed line). Generalised estimating equations, least significant difference posthoc test, n=4–7; #p<0.05, ##p<0.01, ###p<0.001, significantly different from control+vehicle. *p<0.05, **p<0.01, significantly different from post-colitis+vehicle. (C) The effect of combined administration of 10 mg/kg JNJ7777120 and 0.01 mg/kg levocetirizine on colonic compliance in control and postcolitis groups; no significant effect of the factor drug or colitis.

    Discussion

    MCs, as key players of both the innate and adaptive immune system, are powerful neuroimmune modulators implicated in the regulation of various gastrointestinal functions in both physiological and pathophysiological conditions. This study confirms their involvement in the pathogenesis of postinflammatory visceral hypersensitivity and uncovers a substantial contribution of H4Rs. Furthermore, our results confirm the potential role for H1Rs in postinflammatory abdominal pain and reveal a functional interplay between H4Rs and H1Rs.

    Since the discovery of the H4R in 2000, promising results have been published on the effect of H4R antagonists in animal models for acute gastrointestinal inflammation induced by indomethacin, zymosan, TNBS and ischaemia/reperfusion.32 In addition to its anti-inflammatory and immune modulating properties, the selective H4R antagonist JNJ7777120 displayed antiallodynic effects in two models of neuropathic pain—more specifically a L5-L6 spinal nerve ligation and a chronic constriction injury model—pointing towards an additional role for this receptor subtype in nociception.21 We explored the analgesic potential of H4R-targeted therapy in an in vivo model for postinflammatory visceral pain and showed that JNJ7777120 dose-dependently reversed the hypersensitive VMRs to colorectal distension in postcolitis rats, without affecting visceral sensitivity in controls. Importantly, we excluded that this antinociceptive effect was mediated by changes in colonic compliance or by altering the postinflammatory status. To our knowledge, this is the first report demonstrating the involvement of the H4R receptor in visceral hypersensitivity.

    Low to high doses of JNJ7777120 were used in our set-up, ranging from 10 up to 140 mg/kg, based on the compound's pharmacokinetic profile, previous reports in the literature and the high affinity of histamine for the H4R compared with the H1R.19 ,21 JNJ7777120 currently serves as the reference compound for H4R antagonists due to its high affinity for the H4R, which is in the nanomolar range (Ki=4.2 nM for rat H4R).16 ,21 In radio-ligand binding assays, JNJ7777120 displayed very high selectivity over the H1R (Ki >3000 nM), H2R (Ki >3000 nM) and H3R (Ki >1000 nM) as well as over 50 other targets.21 Moreover, in vivo studies administering up to 200 mg/kg confirm the compound's selectivity over the H1R.33 Therefore, we believe that the antinociceptive effects of the doses of JNJ7777120 (10–140 mg/kg) we used are indeed mediated by inhibition of H4Rs.

    In addition, our results demonstrate that levocetirizine, a selective H1R antagonist, also potently modulated postinflammatory visceral hypersensitivity in fully awake rats. These results are in agreement with a report by Stanisor et al34, demonstrating reversal of stress-induced visceral hypersensitivity by fexofenadine, another H1R antagonist, in a rat model of maternal separation. Our data also coincide with a preliminary report that ebastin, yet another selective H1R antagonist, improves abdominal pain in IBS patients.14

    Visceral hypersensitivity can result from peripheral sensitisation of afferent nerves, spinal facilitation through sensitisation of dorsal horn neurons, dysfunction of descending excitatory or inhibitory pathways or from altered central pain processing.35 As H4Rs and H1Rs are expressed on multiple levels of the pain signalling pathways, both peripheral and/or central mechanisms could be involved in the antinociceptive effects of our H4R and H1R antagonists.

    As JNJ7777120 can readily cross the blood–brain barrier, a centrally mediated mechanism is possible.21 Indeed, immunohistochemistry previously revealed the presence of H4Rs in murine spinal cord, cortex, amygdala, hippocampus and thalamus, of which the latter three have been implicated in altered central processing of pain signals in IBS patients.36–39 To date, it remains uncertain whether and to what extent H4Rs are involved in conveying sensory information from the gut to the central nervous system. Previously, histamine-induced jejunal afferent firing in vitro was not affected by a combined H3R/H4R antagonist and in a recent study by Kajihara et al40 JNJ7777120 did not reduce histamine-induced excitation of DRGs.41 However, RT-PCR analysis did reveal expression of the H4R subtype in mice DRGs.39 ,40 Unfortunately none of these studies on DRGs indicate the spinal level at which they were harvested.39 ,40 On the other hand, it remains to be established whether or not these data, gathered in mice models, also pertain to rat colonic afferents. Either way, in our model we were not able to demonstrate H4R mRNA expression in the DRGs containing the pelvic and splanchnic afferent neuronal cell bodies, suggesting the mode of action for JNJ7777120 might not take place at the DRG level. To further elucidate a peripheral mechanism, we evaluated the effects of JNJ7777120 on spontaneous histamine release from the colon. Histamine levels were threefold increased after the resolution of colitis, remained high after an intermediate dose of JNJ7777120 (35 mg/kg) but were no longer different from controls after the highest dose (140 mg/kg). These results suggest a peripheral mechanism for JNJ7777120 that interconnects with the postinflammatory state as the compound impedes excessive histamine release only in the colon of postcolitis rats, probably resulting in a reduction of colorectal distension-induced nociception.

    For the H1R antagonist, a central mechanism of action seems unlikely as levocetirizine shows limited blood–brain barrier penetration.42 In addition, levocetirizine is devoid of central side effects at the clinical daily dose for treatment of allergic rhinitis (5 mg), which is roughly equivalent to the 0.1 mg/kg dose that effectively reduced VMRs in our study.42 Besides it is generally accepted that peripheral histamine stimulates nociceptive nerve fibres via activation of H1Rs.43 In particular for visceral perception, the application of histamine or IBS supernatant containing excessive amounts of MC mediators enhanced mesenteric sensory nerve firing in murine-afferent recordings and stimulated the [Ca2+]i mobilisation in isolated DRGs. These excitatory effects were inhibited by application of a H1R antagonist in all these studies.8 ,41 ,44 The heightened VMRs in our study that were blocked by levocetirizine seem in line with this previous evidence.

    We found arguments for a differential mechanism of action for H1R and H4R antagonists. In contrast to H4R, the colonic H1R mRNA expression level was similar in postinflammatory and control conditions; in addition, H1R but not H4R mRNA was present in the relevant DRGs. These findings seem to concur with the possibility of histamine acting on H1Rs on afferent nerve fibres at the level of the colon and/or the DRG, whereas the activation of H4R seems to occur in the colonic wall, where H4Rs were previously described on intestinal MCs, enteroendocrine cells and immunocytes.17 ,43

    This differential mechanism of action most likely provides the basis for the functional interplay between both receptor subtypes as evidenced by the potentiation of the antinociceptive effect by simultaneous administration of both antagonists. This finding also implies that combination therapy might represent a potent and possibly safe(r) treatment strategy as drug dose reduction most likely results in fewer side effects.

    Our findings undoubtedly corroborate the involvement of histamine in visceral hypersensitivity. Interestingly, similar results have been reported for other MC mediators such as tryptase acting on protease-activated receptors PAR2 and PAR4 and for serotonin, released from enterochromaffin cells located in the gut mucosa.9 ,45–48 This leads to the hypothesis that histamine, tryptase and serotonin might act as sensitising agents modulating a common target. The transient receptor potential vanilloid (TRPV) family is a likely candidate for such a common target.49 In that regard, histamine, tryptase and serotonin have been shown to sensitise TRPV1 and TRPV4 by inducing receptor phosphorylation, translocation and potentiation.49–51 Preliminary data mainly implicate the H1R subtype in this sensitising effect of histamine on TRPV1 and TRPV4, with very little data available on its H4R counterpart, warranting further study.40 ,49

    In summary, considering the emerging role for MCs and their main mediator histamine in the pathogenesis of IBS, we investigated the contribution of H4Rs and H1Rs to postinflammatory visceral hypersensitivity. Both the selective H4R antagonist JNJ7777120 and the selective H1R antagonist levocetirizine normalised increased VMRs to colorectal distension in a rat model for postinflammatory visceral hypersensitivity, with a functional interplay between H4Rs and H1Rs. mRNA expression and, to a lesser extent, histamine release data point towards a different mode of action for both drugs. To conclude, our results reveal that both H4Rs and H1Rs play a substantial role in postinflammatory visceral abdominal pain and bare great potential for specific histamine receptor subtype-targeted therapy in the treatment of postinflammatory visceral hypersensitivity. As H4R antagonists have already entered phase II clinical trials for asthma, clinical studies investigating their effect on IBS symptoms could be initiated in the short term. In addition, our results strongly support the rationale for continuation of clinical trials with H1R antagonists in IBS.

    Acknowledgments

    We would like to thank Janssen Research & Development for supplying JNJ7777120 and Dr R Thurmond for the critical review of the manuscript. A special thanks to our lab technicians, P Aerts, A Jürgens, M Vinckx, A Van Daele and R Van Den Bossche, for their technical assistance.

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    Footnotes

    • Contributors All authors contributed to the conception and design of the study, interpreted the data, revised the manuscript critically for important intellectual content and approved the final version. In addition, AD collected the data, and AD, JGDM and BYDW drafted the manuscript.

    • Funding AD is an aspirant of the Fund for Scientific Research (FWO), Flanders. This work was supported financially by the FWO (G.0341.13 and G.0249.09N).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval Committee for Medical Ethics and the use of Experimental Animals at the University of Antwerp.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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