Histamine H4 Receptor Mediates Chemotaxis and Calcium Mobilization of Mast Cells

TLDR: In this paper, it was shown that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation, and that activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores.

Abstract: The diverse physiological functions of histamine are mediated through distinct histamine receptors. Mast cells are major producers of histamine, yet effects of histamine on mast cells are currently unclear. The present study shows that histamine induces chemotaxis of mouse mast cells, without affecting mast cell degranulation. Mast cell chemotaxis toward histamine could be blocked by the dual H3/H4 receptor antagonist thioperamide, but not by H1 or H2 receptor antagonists. This chemotactic response is mediated by the H4 receptor, because chemotaxis toward histamine was absent in mast cells derived from H4 receptor-deficient mice but was detected in H3 receptor-deficient mast cells. In addition, Northern blot analysis showed the expression of H4 but not H3 receptors on mast cells. Activation of H4 receptors by histamine resulted in calcium mobilization from intracellular calcium stores. Both G alpha i/o proteins and phospholipase C (PLC) are involved in histamine-induced calcium mobilization and chemotaxis in mast cells, because these responses were completely inhibited by pertussis toxin and PLC inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5 (10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). In summary, histamine was shown to mediate signaling and chemotaxis of mast cells via the H4 receptor. This mechanism might be responsible for mast cell accumulation in allergic tissues.

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JPET/2002/46581
1
Histamine H
4
receptor mediates chemotaxis and calcium mobilization of mast cells
Claudia L. Hofstra
1
, Pragnya J. Desai, Robin L. Thurmond and Wai-Ping Fung-Leung.
Johnson and Johnson Pharmaceutical Research and Development L.L.C, San Diego, CA,
USA
Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on March 6, 2003 as DOI:10.1124/jpet.102.046581
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JPET/2002/46581
2
Running title: H
4
receptor mediates chemotaxis and signaling of mast cells
Address of correspondence: Wai-Ping Fung-Leung
Johnson and Johnson Pharmaceutical Research and Development,
3210 Merryfield Row
San Diego, CA 92121, USA
phone: 1-858-450-2016, fax: 1-858-450-2038
wleung@prdus.jnj.com
Number of text pages: 37
Number of tables: 1
Number of figures: 8
Number of references: 36
Number of words in abstract: 189
Number of words in introduction: 765
Number of words in discussion: 1000
Abbreviations:
H
3
R-/- H
3
receptor gene knock-out
H
4
R-/- H
4
receptor gene knock-out
PTX pertussis toxin
Section assignment: Inflammation and Immunopharmacology
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3
Abstract
The diverse physiological functions of histamine are mediated through distinct histamine
receptors. Mast cells are major producers of histamine, yet effects of histamine on mast
cells are currently unclear. The present study shows that histamine induces chemotaxis of
mouse mast cells, without influencing mast cell degranulation. The histamine H
3
and H
4
receptor antagonist thioperamide (but not H
1
or H
2
receptor antagonists) inhibited
histamine-induced chemotaxis of mast cells. The chemotactic response is mediated by H
4
receptors, since chemotaxis toward histamine was absent in mast cells derived from H
4
receptor-deficient mice, but was detected from H
3
receptor-deficient mice. In addition,
Northern blot analysis showed the expression of H
4
but not H
3
receptors on mast cells.
Activation of H
4
receptors by histamine resulted in calcium mobilization derived from
intracellular stores. Calcium mobilization and chemotaxis involve Gαi/o protein
pathways and phospholipase C (PLC), since these responses were completely inhibited
by pertussis toxin (PTX) and PLC inhibitor U73122. In the present study it is shown that
histamine acting on mast cells through the H
4
receptor mediates signaling leading to mast
cell chemotaxis. This mechanism might be responsible for mast cell accumulation in
allergic tissues.
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4
Histamine is a biogenic amine playing an important role in the regulation of different
physiological processes in the body. Histamine is synthesized from L-histidine by
histidine decarboxylation in specific cell types, such as mast cells, basophils,
enterochromaffin like cells and neurons. The diverse biological effects of histamine are
mediated through different histamine receptors, which are all G-protein coupled
receptors. Almost a century of extensive pharmacological research using specific
histamine receptor agonists and antagonists has identified three histamine receptors (H
1
,
H
2
and H
3
receptor). Each receptor has its own expression pattern and mediates distinct
effects: H
1
receptors trigger smooth muscle contractions and is generally thought to play
an important role in allergy; H
2
receptors regulate gastric acid secretion in the stomach
and H
3
receptors control the release of histamine and neurotransmitters by neurons (Hill
et al., 1997). However, not all effects of histamine can be attributed to these three
histamine receptors. Therefore, it has been suggested that another histamine receptor
might exist (Raible et al., 1994). The molecular identity of this fourth human histamine
receptor H
4
receptor) was revealed recently (Oda et al., 2000; Liu et al., 2001a; Morse et
al., 2001; Nguyen et al., 2001; Zhu et al., 2001), and subsequently the H
4
receptor in
mouse, rat and guinea pig were cloned (Liu et al., 2001b).
The amino acid sequence of the H
4
receptor has low homology with other
histamine receptors. Its closest member in the histamine receptor family is the H
3
receptor that shares only a 35% amino acid homology with the H
4
receptor, although the
homology in the transmembrane region is 58%. However, the H
4
receptor expression
pattern is distinct from the H
3
receptor. While the expression of the H
3
receptor is mainly
restricted to cells in the central nervous system (Lovenberg et al., 2000), the H
4
receptor
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seems to be limited to cells of the haemopoietic lineage (Oda et al., 2000; Liu et al.,
2001a; Morse et al., 2001; Zhu et al., 2001). The expression of H
4
receptors on
haemopoietic cells is not unique among the histamine receptor family, since T cells and
dendritic cells also express H
1
and H
2
receptors (Jutel et al., 2001; Szeberenyi et al.,
2001).
Pharmacological properties of the H
4
receptor have been revealed using H
4
receptor-transfected cells (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Nguyen
et al., 2001; Zhu et al., 2001). It was shown that specific H
1
and H
2
receptor antagonists
and agonists do not bind to the H
4
receptor. However, more typical H
3
receptor ligands
(like thioperamide, clobenpropit, imetit and R-α-methylhistamine) could bind the H
4
receptor with affinities different from that of the H
3
receptor.
Similar to other G-protein coupled receptors, histamine receptors activate specific
G proteins that lead to the activation of signal transduction pathways (for review see
(Leurs et al., 1995). It has been shown that H
1
receptors mediate this action through Gαq
proteins resulting in calcium mobilization, H
2
receptors signal through Gαs proteins and
cAMP increase, while H
3
receptors signal through Gαi/o proteins and inhibition of cAMP
(Lovenberg et al., 1999). In literature two signaling pathways are thought to be utilized
by the H
4
receptor. Firstly, using cells transfected with the H
4
receptor and a cAMP
responding reporter construct, studies have shown that histamine could inhibit forskolin-
stimulated cAMP increases (Oda et al., 2000; Liu et al., 2001a; Zhu et al., 2001).
However, this cAMP inhibitory effect is low in comparison with that mediated by the H
3
receptor. Secondly, one study showed that histamine could not alter cAMP levels in H
4
receptor transfected cells, but instead increased calcium mobilization if the cells were
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Frequently asked questions

Q1. What was used as a template to prepare the knockout construct?

A 9 kb mousegenomic fragment containing the mouse H4 receptor gene was obtained from the ES cell line 2G9 and was used as a template to prepare the knockout construct. 

Q2. What is the role of SCF in the calcium mobilization of mast cells?

SCF is also known to induce calcium mobilization in mast cells involving activation of Gαi/o proteins, PI-3 kinase,p38 MAP and MEK kinases (Dastych et al., 1998; Sundstrom et al., 2001). 

Q3. what is the mechanism of calcium release in the cytoplasm?

IP3 can activate an IP3 receptor in the endoplasmic reticulum, which causes the release of calcium in the cytoplasm, a mechanism that is known to occur in mast cells (Pacher et al., 2000). 

Q4. How long did the mast cells incubate with vehicle?

Mast cells from H4R-/- (diamonds) and wild-type (circles) mice were incubated for 15 min with vehicle (black) or 10 µM histamine (white) followed by 30 min incubationwith DNP-HSA. 

Q5. How was the RNA detected from tissues and purified cells prepared?

Detection of mouse H4 receptor expressionRNA from tissues and purified cells was prepared using a RNeasy kit according tothe manufacturers instructions. 

Q6. How did the mice respond to histamine?

Using specific histamine receptor antagonists as well as mast cells derived fromH4R-/- and H3R-/- mice, the authors demonstrated that histamine-induced calcium mobilization from intra-cellular stores in mast cells via the H4 receptor. 

Q7. What is the role of thioperamide in mast cell chemotaxis?

Similar to the PTX inhibitory effects on histamine-induced calcium mobilization,preincubation of mast cells with PTX caused a concentration-dependent decrease in mast cell chemotaxis toward histamine (figure 7D). 

Q8. What is the role of the H4 receptor in the regulation of the immune system?

Each receptor has its own expression pattern and mediates distinct effects: H1 receptors trigger smooth muscle contractions and is generally thought to play an important role in allergy; H2 receptors regulate gastric acid secretion in the stomach and H3 receptors control the release of histamine and neurotransmitters by neurons (Hill et al., 1997). 

Q9. How was the RNA extracted from basophils?

Total RNA was extracted from human basophils, mast cells and HMC1 cells using an RNeasy kit and 250 ng RNA was used for the RT reaction according to manufacturer’s instructions. 

Q10. What is the role of the H4 receptor in the physiological function of mast cells?

In the present study the authors showed that the mouse H4 receptor is expressed on mastcells, and eosinophils, but not on other haemopoietic cells including T cells, B cells or macrophages. 

Q11. What is the role of the H4 receptor in the chemoattractant?

The data presented here shows that histamine is a potent chemoattractant for mast cells and that this chemotaxis is mediated via the H4 receptor. 

Q12. What is the effect of histamine on degranulation of mast cells?

Compound 48/80 induced LTB4 and prostaglandin release by mast cells, whereas histamine did not alter LTB4 or prostaglandin levels (table 1). 

Q13. What was the neomycin resistant gene cassette?

A 0.5 kb regionat A SPE T Journals on A ugust 9, 2022 jpet.aspetjournals.orgD ow nloaded fromJPET/2002/465818covering most of exon 1 and part of intron 1 of the H4 receptor gene was deleted from this genomic fragment and replaced with a neomycin resistant gene cassette.