Histamine H₄ receptors in the gastrointestinal tract

Abstract

Histamine is a well-established mediator involved in a variety of physiological and pathophysiological mechanisms and exerts its effect through activation of four histamine receptors (H₁–H₄). The histamine H₄ receptor is the newest member of this histamine receptor family, and is expressed throughout the gastrointestinal tract as well as in the liver, pancreas and bile ducts. Functional studies using a combination of selective and non-selective H₄ receptor ligands have rapidly increased our knowledge of H₄ receptor involvement in gastrointestinal processes both under physiological conditions and in models of disease. Strong evidence points towards a role for H₄ receptors in the modulation of immune-mediated responses in gut inflammation such as in colitis, ischaemia/reperfusion injury, radiation-induced enteropathy and allergic gut reactions. In addition, data have emerged implicating H₄ receptors in gastrointestinal cancerogenesis, sensory signalling, and visceral pain as well as in gastric ulceration. These studies highlight the potential of H₄ receptor targeted therapy in the treatment of various gastrointestinal disorders such as inflammatory bowel disease, irritable bowel syndrome and cancer.

Tables of Links

TARGETS
GPCRsa
Histamine H1 receptor
Histamine H2 receptor
Histamine H3 receptor
Histamine H4 receptor
Enzymesb
COX-2

LIGANDS
4-Methylhistamine	Immepip
Cimetidine	JNJ10191584
Clobenpropit	JNJ7777120
Clozapine	Ketotifen
Dimaprit	Pyrilamine
Histamine	Thioperamide
Imetit	VUF8430

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (^a,bAlexander et al., 2013a,b).

Introduction

Histamine (2-[4-imidazole]-ethylamine) is a short-acting endogenous amine, involved in several physiological and pathophysiological processes (Jutel et al., 2009). It is present in virtually all bodily organs, with high concentrations reported in the stomach, lymph nodes and thymus (Kumar et al., 1968; Zimmermann et al., 2011). Histamine is synthetized from L-histidine by L-histidine decarboxylase and is stored in the granules of mast cells (MCs) and basophils, the main sources of histamine (Endo, 1982; Jones and Kearns, 2011). Enterochromaffin-like cells, histaminergic neurons, lymphocytes, monocytes, platelets and neutrophils also express L-histidine decarboxylase and are capable of producing, but not storing, high amounts of histamine (Snyder and Epps, 1968; Vanhala et al., 1994; Bencsath et al., 1998; Jutel et al., 2009; Alcaniz et al., 2013). Histamine exerts its actions by binding to four GPCRs that are differentially expressed throughout the body and designated as the H₁, H₂, H₃ and H₄ receptors. Histamine H₁ receptors mediate sensorineural signalling, vascular dilatation and permeability and airway smooth muscle contraction, and are involved in allergic rhinitis, atopic dermatitis, conjunctivitis, urticaria, asthma and anaphylaxis (Togias, 2003; Simons and Simons, 2011). Histamine H₂ receptors are well-known for their role in gastric acid secretion, but also exert immune modulatory properties (Black et al., 1972; Jutel et al., 2009). Histamine H₃ receptors are most abundantly present in the CNS and are implicated in sleep–wake disorders, attention-deficient hyperactivity disorder, epilepsy, cognitive impairment and obesity (Kuhne et al., 2011; Singh and Jadhav, 2013). Finally, histamine H₄ receptors are predominantly expressed on immune cells, such as lymphocytes, MCs and dendritic cells, and are currently mainly under evaluation for immune-mediated disorders such as allergic rhinitis, asthma and pruritus (Liu, 2014). However, new roles for this receptor subtype are continuously being discovered. Here we provide an overview of the current evidence of H₄ receptor involvement in multiple gastrointestinal physiological and pathophysiological processes.

H₄ receptors

In the early 2000s, several groups reported on the discovery and cloning of a fourth histamine receptor (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). The H₄ receptor is encoded by a single copy on chromosome 18q11.2 and demonstrates an overall homology of 23% to H₁ receptors, 22% to H₂ receptors and 37% to H₃ receptors (Oda et al., 2000; Coge et al., 2001). The human full-length receptor consists of 390 amino acids, which form seven transmembrane helices, three extracellular loops and three intracellular loops, with an extracellular N-terminal and an intracellular C-terminal peptide (Leurs et al., 2009). H₄ receptors couple to G_αi/0 proteins, inhibiting downstream adenylyl cyclase and forskolin-induced cAMP (Morse et al., 2001; Zhu et al., 2001). They are mainly present in immune cells and highly expressed in bone marrow and spleen; varying expression levels were also reported in gastrointestinal tissues, testes, kidney, lung, prostrate and brain (Nakamura et al., 2000; Oda et al., 2000; Coge et al., 2001; Strakhova et al., 2009). Tissue distribution is quite similar across species (Liu et al., 2001b; Oda et al., 2005). There is high homology in the amino acid sequence between human and monkey H₄ receptors (92%), whereas this is 72% between human and pig and 65–70% between human and rodent H₄ receptors (Liu et al., 2001b; Oda et al., 2002; 2005,). These differences in amino acid sequence also affect the binding profile of histamine towards H₄ receptors with high affinity for human and guinea pig H₄ receptors (K_D 4.8 and 6 nM) compared with rat and mouse H₄ receptors (136 and 42 nM) (Liu et al., 2001b). Compared with H₁ and H₂ receptors, histamine displays high affinity for H₄ receptors in both human and rodents (Table 2006).

Table 1.

Ligands for the human H₄ receptor

Compound	H4R (pKi)	H1R (pKi)	H2R (pKi)	H3R (pKi)
Agonists
Histamine	7.8	4.2	4.3	8.0
4-methylhistamine	7.3	<5.0	5.1	5.2
Clozapine	6.7	9.4		6.6
Clobenpropit	8.1			8.6
Dimaprit	6.5		4.6	7.3
Imetit	8.2			8.8
Immepip	7.7			9.3
OUP-16	6.9			5.7
VUF10460	8.2			5.8
VUF6884	7.6			5.0
VUF8430	7.5			6.0
Antagonists
A-943931	8.3
JNJ10191584*	7.6
JNJ39758979	7.9	<6	<6	<6
JNJ7777120	7.8	<5.0	<5.0	5.3
ZPL3893787	8.6			6.7
Thioperamide	6.9			7.3
UR-63325	7.8	<5.4	<5.4	<5.4

^*Former VUF6002. Based on Andaloussi et al. (2013); Alfon et al. (2011); Coruzzi et al. (2007; 2011,); Cowart et al. (2008); Leurs et al. (2009); Lim et al. (2009); Mowbray et al. (2011); Oda et al. (2000); Salcedo et al. (2013); Smits et al. (2009); Thurmond et al. (2004; 2014). Of note, ligand affinity may differ among species. Data presented as K_i value (nM) for the human histamine H₄ and H₃ receptors.

Soon after its discovery and cloning, attempts were made to elucidate the pharmacological profile of H₄ receptors and identify (selective) ligands to stimulate or inhibit H₄ receptor signalling. Early assessments indicated that several H₃ receptor ligands demonstrated significant affinity for H₄ receptors, such as clozapine, imetit and immepip (H₃ and H₄ receptor agonists) and clobenpropit (H₃ receptor antagonist, H₄ receptor agonist) (Table 2006) (Leurs et al., 2009; Smits et al., 2009). Since then, several new compounds have been developed targeting H₄ receptors such as 4-methylhistamine, VUF8430 and OUP-16 (selective agonists) and A-943931, JNJ7777120 and VUF6002 (selective antagonists; Table 2006) (Leurs et al., 2009; Smits et al., 2009). However, it was recently demonstrated that in addition to inhibition of Gα_i/0 proteins, many H₄ receptor antagonists can also exert a partial agonist effect at certain species H₄ receptor orthologues via β-arrestin recruitment and ERK activation (Rosethorne and Charlton, 2011), which may contribute to some of the species differences that have been reported for H₄ receptor ligands (Liu et al., 2001b; Seifert et al., 2011; Nijmeijer et al., 2013; Salcedo et al., 2013).

Histamine and histamine receptors in the gastrointestinal tract

In the gastrointestinal tract, histamine participates in multiple physiological processes among which immunological responses, visceral nociception, modulation of intestinal motility and gastric acid secretion (Black et al., 1972; Poli et al., 2001; Dawicki and Marshall, 2007; Takagaki et al., 2009; Simon et al., 2011; van Diest et al., 2012). Histamine is also involved in several gastrointestinal disorders such as inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), malignancies, systemic mastocytosis, food allergy and gastric ulcers (Black et al., 1972; He, 2004; Wood, 2004; Barbara et al., 2006; Sokol et al., 2010; Kennedy et al., 2012). All four histamine receptors are expressed in the gastrointestinal tract, although the presence of H₃ receptors in the human gut remains controversial (Poli et al., 2001). Human H₁ receptors are abundantly expressed throughout the gastrointestinal tract on enterocytes as well as connective tissue cells, immune cells, blood vessels, myocytes and enteric nerves (Sander et al., 2006). H₂ receptors are present on gastric parietal cells, enterocytes, immunocytes such as lymphocytes, myenteric ganglia and smooth muscle cells (Fukushima et al., 1999; Sander et al., 2006). H₃ receptors were reported to be expressed in gastrointestinal tissue of guinea pig and functional data located them on nerve terminals in the myenteric plexus and on pre- and post-ganglionic cholinergic and non-adrenergic, non-cholinergic fibres (Poli et al., 2001). However, human intestine seems to be devoid of H₃ receptors (Hemedah et al., 2001; Poli et al., 2001; Cianchi et al., 2005; Sander et al., 2006).

Using a variety of techniques, several groups demonstrated expression of H₄ receptors throughout the gastrointestinal tract and in the pancreas, liver and bile ducts, not only in humans, but also in other species such as rodents, pigs, dogs and monkeys (Table 2005). Sander et al. (2006) reported similar distribution of H₄ receptors along the human duodenum, colon, sigmoid and rectum. More specifically, H₄ receptors were present on lamina propria mononuclear cells and intestinal MCs, on leucocytes in mucosal and submucosal blood vessels and to a lesser extent on tissue resident leucocytes. In addition, H₄ receptor immunoreactivity was seen in intraepithelial cells considered to be neuroendocrine cells, in myenteric ganglion cell somata and neuronal fibres, and on enterocytes in the crypt of Lieberkühn (Sander et al., 2006; Chazot et al., 2007). Expression of H₄ receptors on colonic enterocytes was later confirmed by others, who also reported limited staining of non-specified submucosal and connective tissue cells (Boer et al., 2008; Fang et al., 2011). A caveat must be made when interpreting data obtained by immunohistochemistry. Recently the selectivity of commercially available H₄ receptor-antibodies was questioned as several of these antibodies failed to yield a specific signal when evaluated in transfected or H₄ receptor^−/− cells (Beermann et al., 2012).

Table 2.

Expression of H₄ receptors in the gastrointestinal tract of different species

Tissue and species	Technique	Expression profile	Reference
Oesophagus
Guinea pig	Immunofluorescence	MCs and eosinophils	Yu et al. (2008)
Stomach
Human	RNase protection assay		Liu et al. (2001a)
	Northern blot		Morse et al. (2001)
	RT-PCR	Mucosa	Zhang et al. (2012)
	Western blot	Mucosa	Zhang et al. (2012)
	Immunofluorescence	Mucosal cells	Zhang et al. (2012)
Rat	Immunohistochemistry	Ganglion cell somata and neuronal fibres in the myenteric but not the submucous plexus; A-like cells in the fundic epithelium	Chazot et al. (2007); Morini et al. (2008)
Duodenum
Human	RT-PCR		Sander et al. (2006)
Small intestine
Human	RNase protection assay		Liu et al. (2001a)
	Northern blot		Morse et al. (2001)
	RT-PCR		Coge et al. (2001); Nakamura et al. (2000); Oda et al. (2000)
Dog	RT-PCR		Jiang et al. (2008)
Rat	Immunohistochemistry	Ganglion cell somata and neuronal fibres in the myenteric plexus	Chazot et al. (2007)
Colon
Human	RNase protection assay		Liu et al. (2001a)
	Northern blot		Morse et al. (2001)
	RT-PCR	Lamina propria mononuclear cells and MCs, mucosa	Boer et al. (2008); Cianchi et al. (2005); Fang et al. (2011); Oda et al. (2000); Sander et al. (2006)
	Western blot	Mucosa	Boer et al. (2008); Fang et al. (2011)
	Immunohistochemistry	Neuroendocrine-like cells, lamina propria, intravascular granulocytes, enterocytes, non-epithelial mucosal cells, submucosal connective tissue cells	Boer et al. (2008); Fang et al. (2011); Sander et al. (2006)
Dog	RT-PCR		Eisenschenk et al. (2011)
Rat	RT-PCR		Deiteren et al. (2014)
	Immunohistochemistry	Ganglion cell somata and neuronal fibres in the myenteric, but not the submucous plexus	Chazot et al. (2007)
Mouse	RT-PCR		Sutton et al. (2008)
Monkey	RT-PCR	Longitudinal muscle	Kim et al. (2011); Oda et al. (2005)
Pig	RT-PCR		Oda et al. (2002)
Pancreas
Human	Northern blot		Morse et al. (2001)
Liver
Human	RNase protection assay		Liu et al. (2001a)
	Northern blot		Morse et al. (2001)
	RT-PCR		Coge et al. (2001); Nakamura et al. (2000)
Dog	RT-PCR		Eisenschenk et al. (2011); Jiang et al. (2008)
Bile ducts
Human	RT-PCR		Francis et al. (2012)
	Western blot		Francis et al. (2012)
	Immunohistochemistry	Cholangiocytes	Francis et al. (2012); Meng et al. (2011)

A caveat must be made when interpreting H₄ receptor expression data obtained by immunohistochemistry: recently the selectivity of commercially available antibodies for the H₄ receptor was questioned as several of these antibodies failed to yield a specific signal when evaluated in transfected or H₄ receptor ^−/− cells (Beermann et al., 2012).

Interestingly, gastrointestinal H₄ receptor expression is altered in several disease states. Decreased H₄ receptor expression was reported in gastric cancer specimens, whereas overexpression was demonstrated in cholangiocarcinoma and both enhanced and decreased expression levels have been reported in colorectal cancer (Cianchi et al., 2005; Boer et al., 2008; Fang et al., 2011; Meng et al., 2011; Francis et al., 2012; Zhang et al., 2012). Colonic inflammation seems to enhance H₄ receptor expression as in two experimental models of IBD, namely murine trinitrobenzene sulphonic acid (TNBS)-induced colitis and spontaneous colitis in G_iα2 protein-deficient mice, active inflammation was associated with an increase in colonic H₄ receptor mRNA (Sutton et al., 2008; Kumawat et al., 2010). Also after complete resolution of TNBS-colitis, colonic H₄ receptor mRNA levels remained increased (Deiteren et al., 2014). However, in colonic biopsies of IBS patients with concomitant food allergy, no alterations in H₄ receptor mRNA levels were reported (Sander et al., 2006).

H₄ receptors and gastrointestinal inflammation

Because of the high levels of expression of H₄ receptors on immunocytes, the immune modulatory potential of this receptor subtype attracted much attention, culminating in clinical trials with H₄ receptor antagonists in immune-mediated disorders such as asthma and allergic rhinitis. Also in the gastrointestinal tract, their immune modulatory properties have been studied using models of colitis, ischaemia/reperfusion injury and allergic gut reactions (Table 2012).

Table 3.

Preclinical in vivo experiments with H₄ receptor ligands in models of inflammation

Model	Species	In vitro/in vivo	Ligand	Effect	Ref
TNBS-induced colitis	Rat	In vivo	JNJ7777120	JNJ7777120 and JNJ10191584 reduced TNBS-induced colitis	Varga et al. (2005)
			JNJ10191584
TNBS-induced colitis	Rat	In vivo	JNJ10191584	JNJ10191584 reduced TNBS-induced colitis	Dunford et al. (2006b)
TNBS-induced colitis	Rat	In vivo	Thioperamide	Thioperamide reduced TNBS-induced colitis	Fogel et al. (2007)
Acetic acid-induced colitis	Rat	In vivo	Thioperamide	Thioperamide reduced acetic acid-induced colitis	Fogel et al. (2005)
Ischaemia/reperfusion intestinal injury	Mouse	In vivo	Thioperamide	Thioperamide reduced reperfusion injury	Ghizzardi et al. (2009)
Ischaemia/reperfusion liver injury	Rat	In vivo	Dimaprit	Histamine, dimaprit and clozapine reduced liver injury	Adachi et al. (2006)
			Clozapine
			Thioperamide	Thioperamide reversed the protective effect of histamine and dimaprit
Ischaemia/reperfusion liver injury	Rat	In vivo	Clozapine	Histamine and clozapine prevented reperfusion injury	El-Mahdy et al. (2013)
			Thioperamide	Thioperamide reversed the protective effect of histamine
Radiation-induced small intestinal damage	Rat	In vivo	JNJ7777120	JNJ7777120 reduced radiation-induced intestinal damage	Martinel Lamas et al. (2013)
Allergen challenge in sensitized oesophagus	Guinea pig	In vivo	Thioperamide	Thioperamide inhibited MC and eosinophil migration	Yu et al. (2008)

Clozapine, H₃ and H₄ receptor agonist; dimaprit, H₂ and H₄ receptor agonist; JNJ10191584, H₄ receptor antagonist; JNJ7777120, H₄ receptor antagonist; thioperamide, H₃ and H₄ receptor antagonist.

MCs, an important source of gastrointestinal histamine, are key players of both the innate and adaptive immune systems and congregate at the interface between the internal and external milieu (such as the gut mucosa), where they exert immune modulatory effects. Alterations in MC numbers and activation state with excessive release of histamine have been reported in patients with IBD (Knutson et al., 1990; Bischoff et al., 1996; Farhadi et al., 2007). Moreover, treatment with the MC stabilizer ketotifen prevented chemically induced colitis in animal models and improved disease activity in a small group of IBD patients; however, the underlying mechanism of action was not investigated further (Eliakim et al., 1992; Jones et al., 1998; Marshall and Irvine, 1998; Fogel et al., 2005). Ketotifen stabilizes MCs (in addition to H₁ receptor antagonist properties), and thus inhibits the release of histamine in the gut; this may indirectly beneficially affect H₄ receptor-mediated pathways activated by histamine.

The selective H₄ receptor antagonists JNJ7777120 and JNJ10191584 and the H₃/H₄ receptor antagonist thioperamide also reduced chemically induced colitis in different rat models for IBD (Fogel et al., 2005; 2007,; Varga et al., 2005; Dunford et al., 2006b). More specifically, treatment with these antagonists reduced macroscopic colonic injury, neutrophil influx and myeloperoxidase levels (a marker for myeloid cell infiltration) (Fogel et al., 2005; 2007,; Varga et al., 2005; Dunford et al., 2006b). This is in line with previous evidence demonstrating that blockade of H₄ receptors impedes neutrophil recruitment and cytokine release in other models of inflammation, such as zymosan-induced pleuritis and allergic airway inflammation (Takeshita et al., 2003; Thurmond et al., 2004; Dunford et al., 2006a). The anti-inflammatory effect of H₄ receptor antagonism resulted – at least partly – from inhibition of aberrant Toll-like receptor signalling via dendritic cells leading to reduced production of TNF-α and IL-6 (Fogel et al., 2005; Varga et al., 2005; Dunford et al., 2006b). In addition, colonic H₄ receptor expression was reported to be increased in the colon of mice with TNBS-induced colitis and during spontaneous colitis in Gα_i2 protein-deficient mice (Sutton et al., 2008; Kumawat et al., 2010). Whether H₄ receptor expression is also increased in IBD patients is an interesting question that has not been investigated to our knowledge.

Data have also emerged, suggesting a possible role for H₄ receptors in mediating gastrointestinal inflammation in ischaemia/reperfusion models. However, in most of these studies non-selective antagonists were used, making it difficult to ascertain that this effect was indeed solely mediated by the H₄ receptor subtype. In a mouse model of mesenteric ischaemia/reperfusion injury treatment with the H₃/H₄ receptor antagonist thioperamide significantly reduced myeloperoxidase activity (Ghizzardi et al., 2009). In contrast, the opposite effect was seen on hepatic ischaemia/reperfusion damage: histamine, the H₂/H₄ receptor agonist dimaprit and the H₃/H₄ receptor agonist clozapine reduced post-ischemic liver damage, as shown by a reduction in serum transaminases (Adachi et al., 2006). This protective effect was abolished by the H₃/H₄ receptor antagonist thioperamide but remained unaffected by the selective H₂ receptor antagonist cimetidine, suggesting a beneficial influence of H₄ receptor stimulation in the prevention of ischaemia/reperfusion liver damage. Recently, the mechanism of action was further elucidated by El-Mahdy et al. (2013). They found that liver damage was significantly reduced by pretreatment with histamine, remained unaffected by a selective H₁ or H₂ receptor antagonist, was abolished by the H₃/H₄ receptor antagonist thioperamide and was reproduced by the H₃/H₄ receptor agonist clozapine. The protective effect of histamine and clozapine was mediated by attenuating TNF-α and IL-12 secretion and consequently reduced reactive oxygen species (El-Mahdy et al., 2013). As H₃ receptors were absent from adult mouse liver tissue (Heron et al., 2001), it seems reasonable to assume that the protective effect of histamine and clozapine was indeed mediated by H₄ receptors. However, it is important to exclude the possibility that hepatic ischaemia/reperfusion does not induce H₃ receptor expression to be sure that the effect is due to H₄ receptor modulation.

Pronounced gastrointestinal inflammation is seen after radiation and results from reactive oxygen/nitrate species, apoptosis and clonogenic cell death, mucosal breakdown and transcription of proinflammatory cytokines, chemokines and growth factors (Francois et al., 2013). In view of the promising results of H₄ receptor blockade on gastrointestinal inflammation in other animal models, Martinel Lamas et al. (2013) evaluated the radioprotective potential of JNJ7777120, a selective H₄ receptor antagonist. Preventive treatment with JNJ7777120 preserved the villi and the number of crypts in the small intestine and diminished mucosal atrophy after radiation by reducing apoptosis and DNA damage in enterocytes (Martinel Lamas et al., 2013).

Finally, preliminary evidence also points towards a possible involvement of H₄ receptors in allergic gut reactions (Yu et al., 2008). Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin; MC and eosinophil infiltration into the oesophagus was assessed 1 h later. Pretreatment with the H₃/H₄ receptor antagonist thioperamide inhibited migration of both cell types to the oesophageal epithelium (Yu et al., 2008). As both MCs and eosinophils did not express H₃ receptors, the effect was ascribed to blockade of H₄ receptors, which seems consistent with previous reports of H₄ receptor-mediated chemotaxis of these cell types (Hofstra et al., 2003; Thurmond et al., 2004; Yu et al., 2008).

In conclusion, these in vivo experiments suggest that H₄ receptors participate in mediating gastrointestinal inflammation and immune responses in a variety of animal models. These findings are in line with previous preclinical observations from immune-mediated disorders in other organ systems and underline the immunomodulatory role of H₄ receptors. However, further research confirming these findings using highly selective ligands for H₄ receptors are much needed before clinical trials can be initiated for gastrointestinal inflammation and immune-mediated disorders.

H₄ receptors and carcinogenesis

Enhanced expression of L-histidine decarboxylase and high histamine producing and secreting capabilities have been reported in malignancies, such as melanoma, breast, colorectal and pancreatic carcinoma both in experimental models and in human tumour biopsies (Medina and Rivera, 2010; Kennedy et al., 2012). Histamine, released by the malignant cells themselves or by other histamine-secreting cells in the environment such as MCs, acts as a growth factor in an autocrine or paracrine fashion, regulating angiogenesis, cell invasion, migration, differentiation, apoptosis and immune suppression (Medina and Rivera, 2010). These results suggest an important role for histamine in tumour development and progression. Histamine-induced cell proliferation seems to be mediated via H₂ receptors as antagonists for these receptors induced apoptosis in human colorectal and gastric cancer cell lines and in experimental models (Rajendra et al., 2004; Jiang et al., 2010). These findings culminated in clinical trials evaluating the effect of H₂ receptor-targeted therapy in colorectal cancer, indicating a beneficial effect when H₂ receptor antagonists were given as therapy, adjuvant to curative surgical resection (Deva and Jameson, 2012). Interestingly, H₂ receptor expression was comparable in colorectal cancer and adjacent normal mucosal specimens, whereas H₁ receptor and H₄ receptor expression were significantly reduced in tumour tissue (Boer et al., 2008; Fang et al., 2011). These findings suggest that carcinogenesis might benefit from loss of H₄ receptors (and H₁ receptors). A potential antiproliferative action of H₄ receptors in colorectal cancer was further substantiated by in vitro experiments demonstrating that stimulation of H₄ receptors induced a cell cycle arrest in the G1 phase via a cAMP-dependent pathway, resulting in reduced cell proliferation and tumour growth (Table 2013) (Fang et al., 2011). This antiproliferative action was only present in H₄ receptor-expressing colorectal cancer cell lines, but not in mock-transfected cells and could be prevented by pretreatment with the selective H₄ receptor antagonist JNJ7777120, further corroborating involvement of these receptors. In addition, H₄ receptor stimulation enhanced apoptosis induced by the chemotherapeutic agent 5-fluorouracil (Fang et al., 2011). In contrast, Cianchi et al. (2005) found that H₄ receptor expression was increased in colorectal cancer specimens. Moreover, histamine-exposure stimulated cell proliferation and VEGF levels, which were reduced by the H₄ receptor antagonist JNJ7777120 (and the H₂ receptor antagonist cimetidine). This proliferative effect of H₄ receptor stimulation was mediated by COX 2-induced PGE₂ as it was only evident in those cell lines that expressed COX 2 (Cianchi et al., 2005). In addition, JNJ7777120 only reduced histamine-induced cell proliferation, but did not affect basal (non-histamine stimulated) cell growth (Coruzzi et al., 2012).

Table 4.

Preclinical in vitro and in vivo experiments with H₄ receptor ligands on carcinogenesis

Model	Species	In vitro/in vivo	Ligand	Effect	Ref
Colorectal cancer cell line	Human	In vitro	Clozapine	Clozapine and clobenpropit reduced cell growth	Fang et al. (2011)
			Clobenpropit	Clozapine enhanced 5-FU induced apoptosis, which was reversed by JNJ7777120
			JNJ7777120
Colorectal cancer cell line	Human	In vitro	JNJ7777120	JNJ7777120 prevented histamine-induced COX-2 expression/activity, cell proliferation and VEGF production	Cianchi et al. (2005)
Gastric cancer cell line	Human	In vitro	Clobenpropit	JNJ7777120 abolished clobenpropit-induced cell growth	Zhang et al. (2012)
			JNJ7777120
Pancreatic duct carcinoma cell line	Human	In vitro	Clobenpropit	Clobenpropit stimulation reduces cell growth	Cricco et al. (2008)
Cholangiocarcinoma cell line	Human	In vitro	Clobenpropit	Clobenpropit inhibited cell proliferation and metastatic potential	Meng et al. (2011)
Cholangiocarcinoma cell line	Human	In vitro	Thioperamide	No effect on histamine secretion and cell growth	Francis et al. (2012)
Xenograft cholangiocarcinoma	Mouse	In vivo	Clobenpropit	Clobenpropit inhibited tumour growth	Meng et al. (2011)

5-FU, 5-fluorouracil; clobenpropit, H₃ receptor antagonist, H₄ receptor agonist; clozapine, H₃ and H₄ receptor agonist; JNJ7777120, H₄ receptor antagonist; thioperamide, H₃ and H₄ receptor antagonist.

Attenuated H₄ receptor expression was reported in human gastric cancer specimens and was most prominent in advanced malignancies (Zhang et al., 2012). Similarly to what was previously demonstrated in colorectal cancer, reduced H₄ receptor expression was linked to enhanced cell proliferation as H₄ receptor stimulation with clobenpropit and histamine reduced the growth of gastric cancer cells (Zhang et al., 2012). Although neither ligand is an exclusive H₄ receptor agonist, the involvement of H₄ receptors was inferred from the fact that pretreatment with the selective H₄ receptor antagonist JNJ7777120 completely abolished agonist-induced responses (Zhang et al., 2012). In line with this, clobenpropit reduced tumour cell proliferation in a pancreatic duct carcinoma cell line (Cricco et al., 2008).

In contrast, H₄ receptor expression was enhanced in malignant cholangiocytes from patients with proven cholangiocarcinoma (Meng et al., 2011). H₄ receptor stimulation with the H₃ receptor antagonist/H₄ receptor agonist clobenpropit dose-dependently reduced proliferation of several cholangiocarcinoma cell lines in vitro (Meng et al., 2011). This cytostatic effect resulted from reduced growth potential and disruption of the invading capacity of the cells. As the effect of clobenpropit was maintained in in vitro experiments in which H₃ receptors were knocked down, this indicates that the effects were indeed mediated via H₄ receptors. Importantly, in an elegant in vivo design, the authors demonstrated the clinical potential of H₄ receptor-modulation in this tumour type as treatment with clobenpropit inhibited tumour growth and disrupted its invasive potential in a xenographic cholangiocarcinoma mouse model (Meng et al., 2011). However, in another study, inhibition of H₄ receptors by the H₃/H₄ receptor antagonist thioperamide did not affect cholangiocarcinoma cell line proliferation (Francis et al., 2012).

Overall, these findings indicate that depending on the type of tumour (gastric vs. colorectal vs. cholangiocarcinoma) H₄ receptor-expression can either be decreased or enhanced. It is unclear whether H₄ receptor expression differs in early versus advanced stages, and this would be interesting to investigate further. In addition, although the data gathered from in vitro experiments using different cell lines strongly indicate that H₄ receptors can potently modulate tumour growth and progression, the results are not univocal. To complement these in vitro findings and increase our understanding of the role of H₄ receptors in gastrointestinal carcinogenesis, additional research and in vivo experiments using selective ligands seem crucial.

H₄ receptors and visceral sensory signalling

Visceral hypersensitivity refers to an enhanced perception of stimuli originating from the internal organs and is believed to contribute to abdominal pain in multiple gastrointestinal disorders among which IBD, IBS and functional dyspepsia (Vermeulen et al., 2014). Sensitization of afferent nerve endings in the gut wall is thought to underlie visceral hypersensitivity (Anand et al., 2007). Several lines of evidence indicate that histamine is involved in this process (Buhner and Schemann, 2012; van Diest et al., 2012). For instance, supernatant from IBS colonic biopsies contains increased levels of histamine (Barbara et al., 2007). When applied to human submucous neurons, this supernatant increased neuronal activity and the degree of activation correlated with histamine levels in the supernatant (Buhner et al., 2009). In addition, histamine induced murine jejunal afferent firing and excited primary sensory neurons (Kreis et al., 1998; Brunsden and Grundy, 1999). The pro-nociceptive effect of histamine seems to be mediated – at least partially – by H₁ receptors expressed on sensory afferents, which is consistent with the finding that excitation of rat jejunal afferents by IBS supernatant can be reduced by application of the H₁ receptor antagonist pyrilamine (Barbara et al., 2007). In addition, a role for H₄ receptors in mediating visceral sensory signalling and nociception has emerged (Table 2013a).

Table 5.

Preclinical in vitro and in vivo experiments with H₄ receptor ligands in visceral sensory signalling and nociception

Model	Species	In vitro/in vivo	Ligand	Effect	Ref
Submucous plexus neurons	Human	In vitro	4-methylhistamine	4-methylhistamine-induced excitation reduced by JNJ7777120	Breunig et al. (2007)
			JNJ7777120
Jejunal afferent firing	Rat	In vitro	Thioperamide	Thioperamide reduced histamine-induced jejunal afferent firing	Brunsden and Grundy (1999)
Jejunal afferent firing	Rat	In vivo	Thioperamide	No effect on histamine-induced jejunal afferent firing	Kreis et al. (1998)
Post-inflammatory visceral hypersensitivity	Rat	In vivo	JNJ7777120	JNJ7777120 dose-dependently reversed visceral hypersensitivity	Deiteren et al. (2014)

4-methylhistamine, H₄ receptor agonist; JNJ7777120, H₄ receptor antagonist; thioperamide, H₃ and H₄ receptor antagonist.

Breunig et al. (2007) reported that the H₄ receptor agonist 4-methylhistamine excited human submucous plexus neurons, an effect that was inhibited by the selective H₄ receptor antagonist JNJ7777120. Also, in vitro jejunal afferent excitation by histamine was reversed by the H₃/H₄ receptor antagonist thioperamide (Brunsden and Grundy, 1999), although these results are in contrast to earlier reports in a similar set-up (Kreis et al., 1998). Recently, our group provided in vivo evidence of reduced visceral nociception after blockade of H₄ receptors. Post-inflammatory visceral hypersensitivity was dose-dependently reduced by JNJ7777120 in a rat model of post-inflammatory IBS, without affecting visceral sensitivity in healthy controls (Deiteren et al., 2014). Although increased colonic expression of H₄ receptor mRNA in hypersensitive rats points towards a peripheral mechanism of action, it remains to be determined whether the antinociceptive effect is mediated by blockade of H₄ receptors on sensory afferents directly or indirectly by modulation of H₄ receptors expressed elsewhere in the gut wall (Deiteren et al., 2014). Nevertheless, these findings coincide with previous reports of antinociceptive and analgesic effects of H₄ receptor antagonists in models of somatic and neuropathic pain (independent of their anti-inflammatory properties) (Coruzzi et al., 2007; Hsieh et al., 2010) and emphasize that H₄ receptors are also attractive targets in the modulation of visceral pain.

H₄ receptors and intestinal contractility

Histaminergic control of gastrointestinal contractility and motility is complex and involves all histamine receptor subtypes. H₁ receptors, located in smooth muscle cells, contribute to contractility by increasing calcium availability at the sarcoplasmic level whereas H₂ receptors mainly facilitate cholinergic and non-cholinergic excitatory transmission in intramural neurons (Poli et al., 2001). Although H₃ receptors inhibit the release of excitatory and inhibitory neurotransmitters from the myenteric plexus, their involvement in enteric peristalsis remains unclear, as no effect of H₃ receptor ligands on gastrointestinal transit was seen in in vivo models and the presence of H₃ receptors in the human digestive tract remains controversial (Hemedah et al., 2001; Poli et al., 2001; Cianchi et al., 2005; Sander et al., 2006). Recently, H₄ receptors were reported to be present on murine myenteric neurons (Chazot et al., 2007). In addition, 4-methylhistamine excited human submucous plexus neurons, which could be blocked by the H₄ receptor antagonist JNJ7777120 (Breunig et al., 2007). As the enteric plexus in highly involved in the regulation of reflex behaviour, peristalsis and intestinal secretion, these findings suggest that H₄ receptors could be involved in gut motility and transit (Table 2013b). However, the H₄ receptor agonist VUF8430 did not affect twitch responses induced by electrical field stimulation in rat duodenum (Pozzoli et al., 2009). In addition, no effect was seen from H₄ receptor stimulation on the membrane potential of murine small intestinal interstitial cells of Cajal, the enteric pacemaker cells and conductors of electrical slow waves in intestinal smooth muscle (Kim et al., 2013). In a recent study, longitudinal smooth muscle preparations with an intact myenteric plexus were harvested from guinea pig ileum and exposed to supernatants prepared from colonic biopsies from IBS patients. This supernatant enhanced cholinergic twitch contractions; however, the responses were not affected by a mixture containing antagonists for H₁–H₄ receptors (Balestra et al., 2012). The H₄ receptor agonist 4-methylhistamine increased contractile forces only in longitudinal smooth muscle strips of monkey colon (Kim et al., 2011). However, as these effects were only present when high doses were used, these results need to be interpreted with caution.

Table 6.

Preclinical in vitro experiments with H₄ receptor ligands in gastrointestinal contractility and transit

Model	Species	In vitro/in vivo	Ligand	Effect	Ref
Submucous plexus neurons	Human	In vitro	4-methylhistamine	4-methylhistamine-induced excitation reduced by JNJ7777120	Breunig et al. (2007)
			JNJ7777120
Whole mount duodenum segments	Rat	In vitro	VUF8430	No effect on contractions	Pozzoli et al. (2009)
Longitudinal smooth muscle incl. myenteric plexus	Guinea pig	In vitro	Thioperamide	No effect on contractions induced by IBS supernatant	Balestra et al. (2012)
Colonic smooth muscle strips	Monkey	In vitro	4-methyl-histamine	4-methylhistamine increased contractile force	Kim et al. (2011)
Cultured small intestine interstitial cells of Cajal	Mouse	In vitro	4-methyl-histamine	No effect on pace maker potentials	Kim et al. (2013)

4-methylhistamine, H₄ receptor agonist; IBS, irritable bowel syndrome; JNJ7777120, H₄ receptor antagonist; thioperamide, H₃ and H₄ receptor antagonist; VUF8430, H₄ receptor agonist.

H₄ receptors and gastric acid secretion and ulceration

Histamine is a potent activator of the acid secreting cells of the stomach (Kopic and Geibel, 2010). Binding of histamine to basolateral H₄ receptors activates adenylyl cyclase resulting in accumulation of cAMP and H⁺ secretion. Before the development of proton pump inhibitors, pharmacological blockade of H₂ receptors was the cornerstone of the treatment of acid-related gastrointestinal disorders (Kopic and Geibel, 2010). In addition to H₂ receptor antagonists, H₃ receptor stimulation also exerted gastroprotective effects via increased mucus production in animal models (Coruzzi et al., 2001; Barocelli and Ballabeni, 2003). The homology between H₃ and H₄ receptors subsequently spurred interest in a possible role for H₄ receptors in gastric acid secretion (Table 2011). Overall, the data gathered to date suggest that H₄ receptors do not participate in gastric acid secretion under physiological conditions as neither H₄ receptor agonists such as VUF8430 and VUF10460 nor H₄ receptor antagonists such as JNJ7777120 and VUF5949 affected basal acid production or the macroscopic appearance of the stomach (Lim et al., 2009; Coruzzi et al., 2011; Adami et al., 2012). However, when the mucosal integrity was compromised such as in models of chemically induced gastric ulceration, damage was significantly enhanced by H₄ receptor stimulation and markedly reduced by its blockade (Adami et al., 2005; 2012,; Coruzzi et al., 2009; 2011,). In addition, enhanced chemically induced mucosal damage by H₄ receptor agonists could be prevented by concomitant H₄ receptor antagonists and vice versa (Coruzzi et al., 2009; 2011,). However, the findings are not fully consistent as the H₄ receptor agonists VUF8430 and VUF10460 had no effect on indomethacin/bethanecol-induced lesions in a mouse model whereas the HCl-induced damage in rats was enhanced by both agonists, and in contrast, indomethacin-induced ulcerations were reduced by VUF10460 (Coruzzi et al., 2009; 2011,; Adami et al., 2012). It was hypothesized that species and strain differences might contribute to the differential effects as JNJ7777120 effectively reduced indomethacin/bethanecol-induced lesions in CD-1, NMRI and BALB/c, but not in C57BL/6J mice (Adami et al., 2012). However, it should be kept in mind that several of the compounds used also display considerable affinity for the H₃ receptor, such as VUF8430 [pK_i for rat H₄ receptors of 6.9 vs. 6.5 for rat H₃ receptors (Lim et al., 2009); Table 2006 ], again underscoring the need for selective H₄ receptor ligands. Although these data seem promising, more research is needed to further elucidate the effect of H₄ receptor modulation on gastric ulcer disease. If a beneficial effect of H₄ receptor blockade on gastric ulceration could be confirmed, this would be a major advantage in terms of drug development, as H₄ receptor antagonists are already under evaluation for their anti-inflammatory and analgesic properties.

Table 7.

Preclinical in vivo experiments with H₄ receptor ligands on gastric acid secretion and ulceration

Model	Species	In vitro/in vivo	Ligand	Effect	Ref
Gastric acid secretion	Rat	In vivo	Dimaprit	Dimaprit potently induced gastric secretion, whereas VUF8430 only marginally increased secretion	Lim et al. (2009)
			VUF8430
			JNJ7777120	Induced gastric acid secretion was not affected by JNJ7777120
Indomethacin/bethanechol-induced gastric ulceration	Mouse	In vivo	JNJ7777120	JNJ7777120 reduces lesions in CD-1, NMRI and BALB/c, but not in C57BL/6J mice.	Adami et al. (2012; Coruzzi et al. (2009)
			VUF10460	No effect of VUF10460 and VUF8430 on gastric lesions
			VUF8430
				VUF10460 abolished the protective effect of JNJ7777120
Indomethacin-induced gastric ulceration	Rat	In vivo	VUF5949	VUF5949 and JNJ7777120 reduced indomethacin-induced lesions	Adami et al. (2005); Coruzzi et al. (2009)
			JNJ7777120
			VUF10460	VUF10460 reduced lesions
			VUF8430	VUF8430 only reduced lesions in the presence of a H2 receptor antagonist
HCl-induced gastric ulceration	Rat	In vivo	Immepip	Immepip, VUF8430 and VUF10460 enhanced HCl-induced gastric lesions	Coruzzi et al. (2011)
			VUF8430
			VUF10460
			JNJ7777120	JNJ7777120 abolished the effect of immepip, but not of VUF8430 and VUF10460

Dimaprit, H₂ receptor agonist, H₄ receptor agonist; Immepip, H₃ and H₄ receptor agonist; JNJ7777120, H₄ receptor antagonist; VUF10460, H₄ receptor agonist; VUF5949, H₄ receptor antagonist; VUF8430, H₄ receptor agonist.

Clinical development

To date, no clinical trials with H₄ receptor ligands have been initiated in the field of gastroenterology. However, several H₄ receptor antagonists have already progressed to phase II clinical trials for immune-mediated disorders such as rheumatoid arthritis, asthma, atopic dermatitis and allergic rhinitis (Table 2007). Currently registered clinical trials (http://clinicaltrials.gov) include compounds from Johnson & Johnson (JNJ39758979 and JNJ38518168), Ziarco Pharma (ZPL3893787) and Palau Pharma (UR-63325).

Table 8.

Current clinical trials with H₄ receptor ligands

Compound	Phase	Population	Status	Trial number
JNJ39758979	I	Healthy volunteers	Completed	NCT01081821
	I	Histamine-induced itch in healthy volunteers	Completed	NCT01068223
	I	Rheumatoid arthritis	Completed	NCT01442545
	II	Asthma	Completed	NCT00946569
	II	Atopic dermatitis	Terminated†	NCT01497119
	II	Rheumatoid arthritis	Terminated‡	NCT01480388
	II	Asthma	Withdrawn‡	NCT01493882
JNJ38518168	I	Healthy volunteers	Completed	NCT01442532
	I	Patients with normal or mild to moderate hepatic impairment	Completed	NCT01863784
	I	Healthy volunteers	Completed	NCT01970020
	I	Healthy volunteers on ketonazole	Completed	NCT01690286
	I	Rheumatoid arthritis	Completed	NCT01450982
	II	Rheumatoid arthritis	Recruiting	NCT01862224
	II	Rheumatoid arthritis	Active, not recruiting	NCT01679951
	II	Rheumatoid arthritis	Terminated*	NCT00941707
	II	Asthma	Recruiting	NCT01823016
ZPL3893787§	I	Healthy volunteers	Completed	NCT00992342
	I	Asthma	Completed	NCT00856687
UR63325	II	Allergic rhinitis	Completed	NCT01260753

^*Terminated because of a single, unexpected serious event.

^†Terminated because of two cases of agranulocytosis.

^‡Terminated/withdrawn because of cases of agranulocytosis in trial NCT01497119.

^§Former PF03893787. Clinical trials as registered on http://clinicaltrials.gov on 11 June 2014.

JNJ39758979, derived from the H₄ receptor antagonist JNJ7777120, showed promising results in initial phase I trials, with good pharmacokinetics upon oral dosing with a plasma half-life of 124–157 h after a single oral dose (Thurmond et al., 2014). In addition, the compound was well-tolerated up to 1200 mg in single ascending dose studies and up to 300 mg bid in a multiple ascending dose study; dose-dependent gastrointestinal symptoms were the main adverse events (abnormal faeces, nausea, vomiting and abdominal pain) (Thurmond et al., 2014). A single dose of 600 mg effectively reduced histamine-induced itch in 23 healthy volunteers (Kollmeier et al., 2014). However, a subsequent phase II trial in patients with atopic dermatitis was discontinued due to two cases of drug-induced agranulocytosis, leading to the termination of JNJ39758979 (Thurmond et al., 2014). The agranulocytosis was reported to be related to the chemical structure of JNJ39758979 and not to the H₄ receptor antagonism (Kollmeier et al., 2014; Liu, 2014; Thurmond et al., 2014); further details are expected to be released in the near future (Thurmond et al., 2014). Therefore, the development of other H₄ receptor antagonists is currently being pursued such as JNJ38518168, which has progressed to phase II for rheumatoid arthritis and asthma. However, one of these trials was terminated because of a single, unexpected serious event, which was not specified further. Details on the underlying mechanisms (H₄ receptor-related or compound-specific) are not yet available.

ZPL3893787 (former PF03893787) is a lead compound of Ziarco Pharma and successfully completed phase I single ascending dose and 14 days multiple ascending dose studies in healthy volunteers (Liu, 2014). No results have been published yet; however, Ziarco Pharma communicated on their website that the compound displayed an excellent pharmacokinetic and safety profile. Results from a subsequent proof of concept trial in patients with asthma have not yet been disclosed.

The Palau Pharma compound UR-63325 successfully completed single and multiple dose ascending studies demonstrating a linear pharmacokinetic profile and no safety concerns according to Salcedo et al. (2013); in addition, a phase II clinical trial in allergic rhinitis patients was recently completed and the data are eagerly awaited.

Conclusions

Since their discovery and cloning almost 15 years ago, knowledge on the role of H₄ receptors has increased rapidly. The expression of H₄ receptors on immune cells has spurred interest in H₄ receptor antagonists as a potential new class of anti-inflammatory drugs in the treatment of rheumatoid arthritis and asthma among others. Also in the gastrointestinal tract, there is now strong preclinical evidence that H₄ receptors modulate the inflammatory process, indicating that these receptors could be interesting new targets in the treatment of IBD, ischaemia/reperfusion injury, radiation-induced enteropathy and allergic intestinal reactions. It would be interesting to investigate whether genetic polymorphisms and copy gene number variations for H₄ receptors are linked to gastrointestinal inflammation as was previously reported for other immune-mediated disorders such asthma and atopic dermatitis (Yu et al., 2010; Simon et al., 2012; Chen et al., 2013). In addition, recent data indicate that H₄ receptors also participate in carcinogenesis and gastric ulceration and in mediating IBS-like visceral pain. The preliminary data gathered so far seem promising, but the effects of pharmacological H₄ receptor modulation will need to be confirmed using highly selective ligands, that are devoid of biased signalling and are extensively evaluated in both in vitro and in vivo settings. In addition, the results of ongoing trials with H₄ receptor antagonists for immune-mediated disorders are eagerly awaited and will be crucial for the future of any therapy targeted at H₄ receptors.

Glossary

Abbreviations

IBD

inflammatory bowel disease

IBS

irritable bowel syndrome

MC

mast cell

TNBS

trinitrobenzene sulphonic acid

Conflicts of interest

The authors report no conflicts of interest.