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Histamine, Histamine Receptors, and their Role in Immunomodulation: An Updated Systematic Review

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Histamine, a biological amine, is considered as a principle mediator of many pathological processes regulating several essential events in allergies and autoimmune diseases. It stimulates different biological activities through differential expression of four types of histamine receptors (H1R, H2R, H3R and H4R) on secretion by effector cells (mast cells and basophils) through various immunological or non-immunological stimuli. Since H4R has been discovered very recently and there is paucity of comprehensive literature covering new histamine receptors, their antagonists/agonists, and role in immune regulation and immunomodulation, we tried to update the current aspects and fill the gap in existing literature. This review will highlight the biological and pharmacological characterization of histamine, histamine receptors, their antagonists/agonists, and implications in immune regulation and immunomodulation.
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The Open Immunology Journal, 2009, 2, 9-41 9
1874-2262/09 2009 Bentham Open
Open Access
Histamine, Histamine Receptors, and their Role in Immunomodulation:
An Updated Systematic Review
Mohammad Shahid*,1, Trivendra Tripathi2, Farrukh Sobia1, Shagufta Moin2, Mashiatullah Siddiqui2
and Rahat Ali Khan3
1Section of Immunology and Molecular Biology, Department of Microbiology, 2Department of Biochemistry, and
3Department of Pharmacology, Faculty of Medicine, Jawaharlal Nehru Medical College & Hospital, Aligarh Muslim
University, Aligarh-202002, U.P., India
Abstract: Histamine, a biological amine, is considered as a principle mediator of many pathological processes regulating
several essential events in allergies and autoimmune diseases. It stimulates different biological activities through differen-
tial expression of four types of histamine receptors (H1R, H2R, H3R and H4R) on secretion by effector cells (mast cells
and basophils) through various immunological or non-immunological stimuli. Since H4R has been discovered very re-
cently and there is paucity of comprehensive literature covering new histamine receptors, their antagonists/agonists, and
role in immune regulation and immunomodulation, we tried to update the current aspects and fill the gap in existing litera-
ture. This review will highlight the biological and pharmacological characterization of histamine, histamine receptors,
their antagonists/agonists, and implications in immune regulation and immunomodulation.
Keywords: Histamine, histamine receptors, H4-receptor, antagonists, agonists, immunomodulation.
I. INTRODUCTION
In historical evolution, histamine (biogenic amine) is
probably one of the most important phlogistic ancient media-
tor, and even one of the most intensely studied molecules in
biological systems which have been using histamine,
catecholamines and other chemical mediators to communi-
cate among cells [1]. Histamine was synthesized in 1907 and
characterized in 1910 as a substance (“beta-1”) [2], owing to
its significant competence to constrict guinea pig ileum, and
its cogent vasodepressor action. However, it took 17 years to
demonstr ate its presence in normal tissues [3]. The relation
between histamine and anaphylactic reactions was made
rapidly in 1929, and was identified as a mediator of anaphy-
lactic reactions in 1932 [4, 5], whereas its connection to mast
cells was not made until 1952 [6], and also its connection to
basophils in 1972 [7]. The search for compounds being po-
tent to neutralize the pathological effects of histamine began
at the Pasteur Institute in Paris during the 1930s, and these
compounds were found to partially block the effects of his-
tamine based on the ethylenediamine structure. The first an-
tihistamine compound was the adrenolytic benzodioxan,
piperoxan (933F), reported by Ungar, Parrot and Bovet in
1937 and was shown to block the effect of histamine on the
guinea-pig ileum [8]. It followed shortly by the report of
Bovet and Staub [9] that structurally related to aryl ethers
such as the thymol ether (929F) [8]. The latter antihistamine
compound proved to be highly toxic for clinical develop-
ment; however, the replacement of ether oxygen by an amino
group led to the search of aniline ethylene diamine
*Address correspondence to this author at the Department of Microbiology,
JN Medical College & Hospital, Aligarh Muslim University, Aligarh-202
002, U.P., India; Tel: +91-571-2720382; Fax: +91-571-2720382;
E-mail: shahidsahar@yahoo.co.in; drmohdshahid123@yahoo.com
derivatives. For this noble research on antihistamines and
curare, Bovet was awarded the Nobel Prize in 1957 [8]. It
was being documented that histamine played an important
role as a mediator of allergic reactions indicated by a series
of compounds with antihistamine activity which protected
guinea pigs from anaphylaxis. However, the clinical use of
these compounds in humans was precluded due to their tox-
icity [9]. The first antihistamine, Antergan (phenben-
zamine, RP 2339) was being used in humans [10], but this
compound was subsequently replaced by Neoantergan
(mepyramine, pyrilamine, RP 2786), which is still in use to
counteract the uncomfortable effects of histamine release in
the skin. Many other antihistamines such as diphenhy-
dramine (Benadryl), tripelennamine, chlorpheniramine and
promethazine are also used in similar manner to counteract
the adverse effects of histamine [8]. Subsequently, after
1945, these antihistamines were widely used in the treatment
of various allergic diseases such as hay fever, urticaria, and
allergic rhinitis. However, the side effects were not uncom-
mon and the sedation was a drawback to their use. A very
few side effects were put to their good use; therefore, some
antihistamines such as cyclizine (Marzine) and diphenhy-
dramine in the form of its 8-chlorotheophyllinate (Drama-
mine) are being used as an antiemetics for travel sickness
[8]. By 1950 there were only 20 compounds clinically avail-
able to block the effects of histamine [1], but advances in
histamine receptors (HRs) ligands have ever attracted many
researchers for pharmaceutical developments and are still
highly topical [11].
Histamine (2-(imidazol-4-yl) ethylamine) is one of the
monoamines and was coined after the Greek word for tissue
histos, with the broadest spectrum of activities in various
physiological and pathological conditions including the cell
proliferation, differentiation, hematopoiesis, embryonic de-
velopment, regeneration, wound healing, aminergic neuro-
10 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
transmission and numerous brain functions (sleep/noci-
ception, food intake and aggressive behavior), secretion of
pituitary hormones, regulation of gastrointestinal and circu-
latory functions, cardiovascular system (vasodilatation and
blood pressure reduction), as well as inflammatory reactions,
modulation of the immune response, energy of endocrine
and homeostasis [1, 12-18]. It is being documented by sev-
eral studies which highlighted the evidence of histamine that
it elicits immune-modulatory and pro-inflammatory effects
by the differential expression of histamine receptors (H1R,
H2R, H3R, and H4R) that is easily modulated the diverse
effects of histamine on immune regulation and distinct intra-
cellular signals. All these four receptors are members of the
7-transmembrane (heptahelical) spanning family of recep-
tors, are G protein-coupled (GPCR), are expressed on vari-
ous histamine responsive target tissues and cells and suggest
an important critical role of histamine in immunomodulation
and allergic diseases [1, 8, 11-14].
In the present review, we will discuss biology of hista-
mine including synthesis, regulation and metabolism; hista-
mine receptors including H1-, H2-, H3-, and H4-receptors
and their cellular distribution, functional characterization,
structural biology, and signaling mechanisms; non-classical
histamine-binding sites such as cytochrome P450; and his-
tamine transporters; as well as immune regulation by hista-
mine in immunomodulation and allergic inflammation; ef-
fects of histamine in immune cells in respect to allergic dis-
eases; implication of histamine on cytokines production;
significance of histamine in autoimmunity and allergic dis-
eases and also in malignancies; and finally the relation of
histamine-cytokine during hematopoiesis.
II. BIOLOGY OF HISTAMINE
Histamine exhibits two main important basic functionali-
ties such as primary aliphatic amine (pKa1 9.4) and imidazole
(pKa2 5.8). These make the monocation with different
tautomers; the preferred form at physiologic pH value (96%)
with a minor dicationic fraction (3%) and a very small
amount of the neutral form [19]. The nomenclature for his-
tamine positions may be highly significant for histamine
biology including synthesis, regulation, metabolism, and also
histamine derivatives (Fig. 1).
Fig. (1). Specific nomenclature for histamine positions.
A. Synthesis of Histamine
Histamine was first identified as an auto coid h aving po-
tent vaso active properties. It is a low molecular weight
amine synthesized from L-histidine exclusively by L-
histidine decarboxylase (HDC) (E.C. 4.1.1.22 or E.C.
4.1.1.26), which is dependent on the cofactor pyridoxal-5-
phosphate to a putative binding site (TFNPSKW) on the pro-
tein. Histamine cannot be generated by another enzymatic
pathway [8, 14]. Histidine decarboxylase (HDC) is an en-
zyme that is expressed in various cells through out the body,
including central nervous system, neurons, gastric-mucosa,
parietal cells, mast cells(~3 pg/cell histamine), and baso-
phils(~1 pg/cell histamine). Histamine has an important role
in human health, and exerting its diverse biologic effects by
4 types of receptors [1, 13, 20-22]. Histamine is also pro-
duced by enterochromaffin-like cells (ECL) in the stomach
and plays an important role in secretion of gastric acid [14].
Only basophils and mast cells can store the amine in specific
granules, in the hematopoietic system, where histamine is
closely associated with anionic proteoglycans heparin (in
mast cells) and chondroitin-4-sulfate (in basophils). In this
specific form, histamine can be released in large amounts
during degranulation in response to various immunological
(immunoglobulin E, or cytokines) or non-immunological
(compound 48/80, calcium ionophore, mastoparin, substance
P, opioids, or hypo-osmolar solutions) stimuli [14]. Hista-
mine synthesis in Golgi apparatus can be inhibited by -
fluoromethylhistidin [23].
Recently, many myeloid and lymphoid cell types that do
not store histamine show more HDC activity and are capable
of synthesis of high amounts of histamine [24]. This so
called “neo synthesized histamine,” has been shown in vari-
ous cells, including hematopoietic progenitors, macrophages,
neutrophils, platelets, dendritic cells (DCs) and T cells [14,
25-28]. Histamine synthesis in non-mast cells was first con-
firmed using W/WV mice, which genetically lack mature mast
cells, upon stimulation with a phorbol ester [29]. HDC activ-
ity is demonstrated in vitro through cytokines, such as IL-1,
IL-3, IL-12, IL- 18, GM-CSF, macrophage-colony stimulat-
ing factor, TNF-, and calcium ionophore [30, 31]. Histidine
decarboxylase (HDC) activity has been modulated in condi-
tions such as LPS stimulation, inflammation, infection, and
graft rejection, in vivo [32].
It is being demonstrated that the generation of HDC-
knockout mice provides histamine-free system and it is more
beneficial to study the role of endogenous histamine in a
broad range of normal and disease processes. Such mice
demonstrate diminished numbers of mast cells and signifi-
cantly decreased granule content, which suggests that hista-
mine might affect the production of mast cell granule pro-
teins [33]. In a recent study, interleukin-3 (IL-3)-dependent
bone marrow derived mast cells (BMMCs) have been found
to be activated by certain immunoglobulin-E (IgE) clones in
absence of specific antigen, leading to their survival, cyto-
kine secretion, histamine production, adhesion, and migra-
tion [34]. In addition to this study, Tanaka et al. [35] has
shown a drastic and transient induction of HDC (~ 200-fold
in activity) in BMMCs stimulated by IgE alone, which was
found much higher than that upon antigen stimulation. Thus,
this induction resulted in the increase in stored histamine.
Another study suggested that the anti-apoptic effects of
monomeric IgE on BMMCs were mediated by interleukin-3
(IL-3) in an autocrine fashion [36]. Although Schneider et al.
[30] found the potential role of IL-3 to induce HDC in bone
marrow cells, it is clearly indicated that monomeric IgE-
induced histamine synthesis may not be mediated through
IL-3 [36]. Since stimulation of histamine synthesis occurs
upon IgE-mediated antigen induction, and this remains con-
troversial if these two modes of FcRI activation share a
common signal transduction pathway. However, many recent
studies have demonstrated the qualitative differences be-
tween both modes: such as monomeric IgE-induced Ca2+
influx is mediated by a distinct channel from that activated
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 11
upon antigen stimulation [37], and protein kinase C beta-II
(PKCII) plays a significant role in monomeric IgE-induced
histamine synthesis in mast cells, but not upon antigen
stimulation [38]. Since, only small levels of increase in his-
tamine synthesis were found by monomeric IgE both in puri-
fied rat peritoneal mast cells and in vitro maturated BMMCs,
inducing effects of monomeric IgE on mast cells may be
limited to immature mast cells [37]. However, Tanaka and
Ichikawa [39] has suggested that monomeric IgE-induced
histamine synthesis exacerbates the sympto ms of chronic
allergy, while drastic increases in the levels of serum IgE are
often observed in such diseases.
B. Regulation of Histamine
Histamine is synthesized only by HDC enzyme. There-
fore, histamine regulation is dependent on the gene of HDC
enzyme, which is expressed in the cells throughout the body.
It has been shown that complementary deoxyribonucleic
acids (cDNAs) of HDC enzyme have been isolated from
mouse mastocytoma, fetal rat liver, erythroleukemia cells
and human basophil leukemia cells. Based on structural stud-
ies, mouse and human genes are composed of 12 exons
spanning nearly 24 kb. The 2.4 kb single transcript is pro-
duced by mouse gene, whereas two splice variants of 3.4 kb
and 2.4 kb exist in humans, and latter encode the functional
HDC [40]. HDC gene is found on chromosome 2 in mice
and chromosome 15 in human and their expression is con-
trolled by lineage-specific transcription factors. These factors
interact with a promoter region consisting of GC box, four
GATA consensus sequences, a c-Myb-binding motif and
four CACC boxes [41]. It has been demonstrated in several
studies that the HDC transcription is regulated by various
factors in gastric cancer cells such as gastrin, oxidative stress
and phorbol 12-myristate 13-acetate (PMA), through a Ras-
independent, Raf-dependent mechanism, MAP kinase/ERK
and a protein kinase C (PKC) pathways functioning on three
overlapping cisacting elements (GASRE 1, GAS-RE 2 and
GAS-RE3) known as gastrin response elements [42, 43]. The
negative control on HDC expression in gastric epithelial cell
line is exerted by expression of the transcription factors
GATA-4 and GATA-6 [44]. It is well known that the expres-
sion of HDC in basophils and mast cells seems to be a con-
sequence of the state of CpG methylation in the promoter
region [45]. Many studies on the mast cell line HMC1 and
the pluripotent hematopoietic cell line UT7D1 have demon-
strated that HDC-gene expression is subject to post-
transcriptional control. Therefore, the chromosomal configu-
ration and methylation of the HDC-promoter is likely to ac-
count for its cell-specific expression [46, 47]. It has also
been reported that PMA stimulates a strong increase in HDC
activity in UT7D1, which is affected by actinomycin D, and
that is not paralleled by enhanced HDC mRNA expression.
Similar effect was noted in cell lines (HEL and CMK) with
megakaryocyte/basophil differentiation potential [48]. In
addition to this effect, a mechanism that accounts for the
strong enhancement of HDC activity in ECL cells in re-
sponse to gastrin is explained by a translation control of
HDC expression [49]. Two essential mechanisms of transla-
tional control have been explained in hematopoietic cells: (i)
a rapamycin dependent pathaway that is linked to phosphoi-
nositide 3-kinase (PI3K), FRAP/mTOR and phosphoryla-
tion/dephosphorylation of repressor of translation 4E-
binding protein (4E-Bps) and (ii) ERK- and p38-dependent
pathway that control the 4E-BP expression by the induction
of Egr-1 [50]. The multiple carboxy-truncated isoforms are
formed due to post-translational processing of HDC gene;
the gene is initially translated 73-74 kDa protein in mam-
mals, and originally it was assumed that enzymes purified
from native sources corresponded to a dimer of two proc-
essed isoforms of 53 and 55 kDa. According to Fleming and
Wang [51], the biosynthesis of histamine involves primarily
the 55 kDa isoform and it is being acknowledged that many
other isoforms generated from 74 kDa primary translation
product can also be active. It is also being documented that
enhancing the histidine decarboxylase activity might cause
reduction in messenger RNA (mRNA) degradation by amino
acid carboxyl-terminal PEST domains [52]. Here is a need to
completely understand the negative feed back regulation of
histidine decarboxylase activity that differs from one cell
type to another. This activity has been shown in AGS-B cells
that over expression of the HDC protein inhibited histidine
decarboxylase promoter activity by down regulation of ERK
signals [53].
However, in gastrin-stimulated ECL cells, this type of
feed back mechanism was not observed. It was also demon-
strated that in the hematopoietic cells, as well as in the stom-
ach, negative feed back signals could be produced through
high cytosolic histamine concentration [50]. Histamine reup-
take mechanism comparable to that of the o ther aminergic
neurotransmitters has not been observed [54].
C. Metabolism of Histamine
It is noteworthy that only a small amount of released his-
tamine (2 to 3%) is excreted unchanged. The remaining his-
tamine (more than 97%) is controlled via two major path-
ways for the metabolizing enzymes: histamine N
-
methyltransferase (HMT) (EC 2.1.1.8) and diamine oxidase
(DAO) (EC1.4.3.6) before excretion [23, 55]. Histamine N
-
methyltr ansferase metabolizes the majority of histamine (50
to 80%) to N-methyl histamine, which is further metabolized
to the primary urinary metabolite M-methylimidazole acetic
acid by monoamine oxidase. Diamine oxidase metabolizes
the histamine (15 to 30%) to imidazole acetic acid [20]. The
study of the former pathway was greatly facilitated by the
availability of a potent and highly specific inhibitor of dia-
mine oxidase, aminoguanidine. HMT appears to be the most
important enzyme contributing to the degradation of hista-
mine in the airways, because blockers of HMT (such as SKF
91488) increase the bronchoconstricting action of histamine
in vitro and in vivo, whereas diamine oxidase inhibition re-
mained uneffected [56]. HMT is expressed in airway epithe-
lial cells and may therefore be responsible for the local me-
tabolism of histamine released from airway mast cells. Me-
chanical removal of airway epithelium enhances the bron-
choconstriction response to histamine in vitro [57-59]; this
might be the result, in part, of loss of the metabolizing en-
zyme. Furthermore, experimental viral infections resulted in
reduced epithelial HMT activ ity in association with in-
creased responsiveness to inhaled histamine [60]. The half-
life of pharmacologically active doses of histamine is less
than 10s in the rat and 20-30s in the dog. In earlier studies,
histamine levels were measured by bioassay, but subse-
quently fluorometric and radio-enzymatic techniques were
employed [8].
12 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
III. HISTAMINE RECEPTORS
Histamine is an important biogenic amine and has multi-
ple effects that are mediated through specific surface recep-
tors on specific target cells. Four types of histamine recep-
tors have now been identified. In 1966, histamine receptors
were first differentiated into H1 and H2 [61], and it was re-
ported that some responses to histamine were inhibited by
low doses of mepyramine (pyrilamine), whereas others were
unsympathetic. In 1999, a third histamine receptor subtype
was cloned and termed as H3 [22]. Subsequently in 2000, the
fourth histamine receptor subtype was reported which was
termed as H4 [21] and introduced a sign ifican t chapter in the
story of histamine effects.
A. Histamine H1-Receptor
1. Cellular Distribution and Functional Characterization
In different mammalian tissues, the study of the distribu-
tion of histamine H1-receptors (H1Rs) has been significantly
helped by the development of specific radioligands for this
subtype. In 1997, [3H]mepyramine a selective radioligand
was developed (Table 1) [62], and since then it has been
used to identify H1-receptors in a wide variety of tissues
such as gastrointestinal tract, central nervous system, air-
ways and vascular smooth muscle cells, mammalian brain,
hepatocytes, nerve cells, endothelial cells, chondrocytes,
monocytes, neutrophils, dendritic cells, T and B lymphocytes
(Table 2), the cardiovascular system and genitourinary sys-
tem, endothelial cells and adrenal medulla in which H1-
receptor mediates diff erent biological properties of allergic
responses such as typical immediate responses of allergic
reaction type I like redness, itching and swelling (“triple re-
sponse”). In many pathological processes of allergy, includ-
ing allergenic rhinitis, atopic dermatitis, conjunctivitis, urti-
caria, asthma, and anaphylaxis, H1-receptors are involved.
The receptors also mediate bronchoconstriction and en-
hanced vascular permeability in the lung [17, 63-65]. It has
been noticed that [3H]mepyramine binds to secondary non-
H1-receptor sites in various tissues and cells [66-70]. In ad-
dition to [3H]mepyramine, which predominantly binds to a
protein homologous with debrisoquine 4-hydroxylase cyto-
chrome P450 in rat liver [71], this nonspecific binding can
be blocked by quinine. This investigation led to the demon-
stration that quinine may be used to block binding to other
lower affinity sites [72], and it was thus proved that all sec-
ondary binding sites for [3H]mepyramine were not sensitive
to inhibition through quinine [69]. Many researchers have
shown that a 38 to 40 kDa protein was isolated from
DDT1MF-2 cells, that binds H1R antagonists with specific
KD values in the μM range, but that was not sensitive to in-
hibition through quinine and also that DDT1MF-2 cells pos-
sess [3H]mepyramine binding sites which have the charac-
teristics of histamine H1-receptors (i.e., KD values in the nM
range) to mediate functional responses, and those were pro-
duced by H1R activation [69, 73, 74]. Other radioligands
that have been demonstrated to study histamine H1-receptors
are [3H]mianserin, [3H]doxepin, [125I]iodobolpyramine,
[125I] iodoazidophenpyramine, and [3H](1)-N-methyl-4-
methyldiphenhydramine [75-80]. [125I]Iodobolpyramine has
been successfully used for autoradiographic localization of
H1Rs in the brain of guinea pig, whereas, it was used with
lower success for localization in rat brain (Table 1) [78, 81].
Slow dissociation of [3H]mepyramine from H1Rs has been
shown at low temperature (i.e., 4°C) and this denotes that
[3H]mepyramine can also be used for autoradiography (Ta-
Table 1. Characterization of Histamine Receptors Agonist, Antagonist and Radioligand
Receptor
Subtypes Agonists with Potency Antagonists with Potency Radioligands with Equilibrium Consta nt
for Dissociation (Kd)
H1
Histamine(100) a, b, Dimethylhistaprodifen
(240)a, Methylhistaprodifen (340)a, Histamine-
trifluoromethyltoluidine (HTMT)c,
2-(3-trifluoromethylphenyl) histamine (128)a, b,
2-Thiazolylethylamine (26)a,
2-Pyridylethylamine (6) a
Mepyramine (pA2 9.4) a,
(+)-Chlorpheniramine (pA2 9.4)a, (-)-
Chlor-pheniramine (pA2 6.7) a, Trans-
triprolidine (pA2 10.0) a, Temelastine
(pA2 9.5)a, Promethazine (pA2 8.9) a,
Diphenhydramine (pA2 9.0)a, Tripelen-
namine (pA2 8.5) a, Chlorpromazine (pA2
8.9) a
[3H]-Mepyramine (Kd 0.8nM: guinea-pig
brain, ileum)a,b,
[125I]-Iodobolpyramine (Kd 0.01nM, guinea-
pig brain)a, [125I]-Iodoazidophen-pyramine
(Kd 0.01nM, guinea-pig cerebellum) a, b
H2
Histamine(100) a, b,
Arpromidine (10230) a, b, Impromidine (4810)a,
b, Sopromidine (740) a, b, Amthamine (150) a, b,
Dimaprit (71) a, b,
4-Methylhistamine (43) a, b
Cimetidine (pA2 6.1) a, Ranitidine (pA2
6.7) a, Famotidine (pA2 7.8) a, Zolanti-
dine (pA2 7.6) a, Mifentidine (pA2 7.6) a,
Titotidine (pA2 7.8) a, Iodoaminopotenti-
dine (pA2 8.6) a
[3H]-Tiotidine (25nM) a, b, [125I]-Iodoamino-
potentidine (Kd 0.3nM) a, b,
[125I]-Iodoazido-potentidine (Kd 10nM) a, b
(all guinea-pig brain membrains)
H3
Histamine(100) a, b,
Imetit (6200) a, b,
Immepip (2457) a, b,
R--methylhistamine (1550) a, b
*Thioperamide (pA2 8.4) a, Iodophen-
propit (pA2 9.6) a,
*Clobenpropit (pA2 9.9) a, Ciproxifan
(pA2 9.3) a, Impentamine (pA2 8.4) a,
GR174737 (pA2 8.1) a,b, Impromidine
(pA2 7.2) a
[3H]-R--methylhistamine (Kd 0.5nM) a, b,
[3H]-N-methylhistamine (Kd 2.0nM) a, b,
[125I]-Iodophenpropit (Kd 0.3nm) a, b, [125I]-
Iodoproxyfan (Kd 0.065nM) a, b,
[3H]-GR168320 (Kd 0.1nM)a, b (all rat cere-
bral cortical membranes in Tris buffer)
H4
Imetit (pA2 8.6)c,
Immepip (pA2 8)c,
*Clobenpropit (pA2 7.9, partial agonist)c,
4-Methylhistamine (pA2 7.3)c
JNJ 10191584 (7.6)c, *Thioperamide
(7.6)c None to date
[11a, 23b, 221c]; *These compounds act as agonist/antagonist for different histamine receptors at variable potencies.
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 13
ble 1) [82, 83]. [125I]Iodoazidophenpyramine (Table 1) is a
very potent H1-receptor antagonist that can bind irreversibly
to H1-receptors following irradiation with ultraviolet light
[79]. The existence of H1Rs in the living human brain has
been proved by specific ligands [11C]Mepyramine and
[11C]doxepin [84, 85]. H1Rs have widely been studied in
blood vessels [86-88], and also in smooth muscles [61, 88,
89]. In smooth muscles, such as the guinea pig ileum, which
freely generate muscle action potentials, modulation of ac-
tion-potential discharge by low concentrations of histamine
is an important mechanism by which tension is increased
[90] and there is evidence that the contractile response to
histamine is produced by inositol 1, 4, 5-triphosphate-
induced mobilization of intracellular calcium (Ca2+) [91, 92].
Its further effects have been seen in non excitable smooth
muscles including airways and vascular smooth muscles, and
the contractile H1R stimulation initially involve mobilization
of calcium (Ca2+) from intracellular stores such as inosito l
phospholipids hydrolysis [93-96]. H1-receptor stimulation
causes various cellular responses in vascular endothelial cells
such as: it is responsible for changes in vascular permeability
as a result of endothelial cell contraction [97, 98]; in synthe-
sis of prostacyclin [99, 100]; in platelet-activating factor
synthesis [99]; in release of Von Willebrand factor [101],
and in nitric oxide release [102].
The study of H1R on human T lymphocytes has been
characterized by use of [125I]iodobolpyramine [103] (see
also Table 1) and is shown to increase (Ca2+)i [104]. It is
being documented that H1R-deficient mice display both
strong systemic T cell and efficient B cell responses to anti-
gen [105]. The relationship of H1Rs to adrenal medulla
which elicit the release of catecholamines has been estab-
lished many years ago [106-108]. Thus, histamine can stimu-
late the release of both adrenaline and noradrenaline [108],
and also induce phosphorylation of the catecholamine bio-
synthesis enzyme tyrosine hydroxylase by a mechanism
which mediates release of intracellular calcium from cultured
bovine adrenal chromaffin cells [109].
The effects of histamine are also seen to elicit the release
of leucine- and methionine- enkephalin [110]. Furthermore,
many investigators have demonstrated a marked increase in
mRNA-encoding proenkephalin A after prolonged exposure
to histamine [110, 111]. Its negative inotropic effects have
been observed in human atrial myocardium and also in
guinea pig ventricle [112, 113].
Genovese et al. [113] suggested that the negative
inotropic response of histamine in human myocardium is
associated with inhibitory effects on heart rate. This can be
unmasked when the positive responses of histamine on the
heart rate, and force of contraction (due to histamine H2-
receptors) are mediated through conjoint administration of
adenosine or adenosine A1-receptor agonists. However, his-
tamine produces a positive inotropic effect in guinea pig left
atria and rabbit papillary muscle by a specific mechanism
which is not related with a rise in adenosine 3c, 5c-cyclic
monophosphate (cAMP) levels [90, 114, 115]. It is being
documented that the distribution of H1Rs in mammalian
brain with higher densities are found in neocortex, hippo-
campus, nucleus accumbent, thalamus, and posterior hypo-
thalamus [90, 116], however, cerebellum and basal ganglia
denotes lower densities [76, 85, 117]. The distribution of
H1Rs in rat and guinea pig is very similar to each other [78,
82, 83, 118]. H1-receptor binding sites and mRNA levels
were overlapped in most areas of brain except in hippocam-
pus and cerebellum in which the inconsistency is mostly to
reflect the presence of exuberance H1Rs in dendrites of py-
ramidal and Purkinje cells [119]. The activation of H1R in-
hibits the firing and hyperpolarization in hippocampal neu-
rons [120] and also an apamine sensitive outward current in
olfactory bulb interneurons [121], and these effects are
mostly generated by intracellular Ca2+ release. However,
Table 2. Characteristics of the Histamine Receptor Subtypes
Characteristics H1-Receptor H2-Receptor H3-Receptor H4-Receptor
a, bReceptor described,
human gene cloned (years) 1966, 1993 1972, 1991 1983, 1999 1994, 2000
aReceptor proteins in hu-
man 487 amino acids, 56 kD 359 amino acids, 40 kD 445 amino acids,70 kD;
splice variants 390 amino acids
a,cChromosomal location in
human 3p25, 3p14-21 5, 5q35.3 20, 20q13.33 18q11.2
bEquilibrium constant for
dissociation (Kd) ~10 mol/L ~ 30 mol/L ~ 10 nmol/L 20-40 nmol/L
aReceptor expression
Widespread, including neu-
rons, smo oth muscle (e.g.,
airways, vascular), and other
types of cells.*
Widespread, including
gastric mucosa parietal
cells, smooth-muscle, heart,
and other types of cells.*
High expression in hista-
minergic neurons, low ex-
pression elsewhere.
High expression in bone
marrow and peripheral
hematopoietic cells, low
expression elsewhere.
cGene Structure Intronless Intronless Three introns Two introns
aG-protein coupling Gq/11 Gs Gi/o Gi/o
a,bActivated intracellular
signals (principal signaling
effector molecules)
Ca2+, cGMP, NF-B,
PLC, phospholipase A2,
and D, cAMP, NOS
cAMP, Ca2+,protein kinese
C, c-fos, phos- pholipase C
Ca2+, MAP kinase;
inhibition of cAMP
Ca2+, MAP kinase;
Inhibition of cAMP
Abbreviations: cAMP = cyclic adenosine monophosphate, cGMP = cyclic guanosine monophosphate, MAP = mitogen-activated protein, NF-B = nuclear factor-B, NOS = nitric
oxide synthase, PLC = phospholipase C.
*Other types of cells: epithelial, endothelial cells, neutrophils, eosinophils, monocytes, dendritic cells, T-cells, B cells, hepatocytes, and chondrocytes.
a[399],
b[1],
c[14].
14 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
H1R excite various notable factors such as vegetative gan-
glia [122], hypothalamic supraoptic [23], brainstem [123],
thalamic [124], and human cortical neurons [125] through a
block of potassium conductance.
The functional characterization of H1R has benefited
from the use of many potent and specific antagonists (see
Tables 1 and 3) [63, 126]. H1-receptor antagonists are the
oldest therapeutic tools of the modern medicine due to their
sedative side effects, and the anti-allergic drugs which were
developed initially, have now been abandoned. Indeed, H1-
receptor involves the disturbance of circadian rhythms and
locomotor activ ities as well as the impairment of the ex-
ploratory behavior by histamine in the brain, and this is why
so-called “non-sedating” H1 antagonists which cannot cross
the blood-brain barrier have been designed. H1-receptor
agonists are not readily available because they enhance
rather than prevent the onset of allergic pathologies. His-
taprodifens are very potent H1R agonists and are more effec-
tive than histamine in activating H1R [127]. Some anti-
inflammatory effects of H1R antagonists at high doses could
be non-specific because of histamine and other inflammatory
mediators like leukotriene and platelet activating factors re-
leased from basophils in response to certain H1Rs antago-
nists [1, 14]. Bordetella pertusis-induced histamine sensitiza-
tion (Bphs) controls Bordetella pertussis toxin (PTX)-
induced vasoactive amine sensitization elicited by histamine
(VAASH) and has an established role in autoimmunity. The
congenic mapping links Bphs to the histamine H1 receptor
gene (Hrh1/H1R) and that H1R differs at three amino acid
residues in VAASH-susceptible and -resistant mice. Hrh1-/-
mice are protected from VAASH, which can be restored by
genetic complementation with a susceptible Bphs/Hrh1 al-
lele, and experimental allergic encephalomyelitis and auto-
immune orchitis due to immune deviation. Thus, natural al-
leles of Hrh1 control both the autoimmune T cells and vascu-
lar responses regulated by histamine after PTX sensitization.
The exact mechanism through which this effect occurs re-
mains unclear and its clinical relevance is still uncertain
[128]. The chemical structure of specific H1R-antagonists
and agonists are shown in Figs. (2, 3).
2. Structural Biology of Receptor
H
1 receptors have been cloned from cows, rats, guinea
pigs and also from humans. The H1 receptor contains 486,
488 or 487 amino acids in rat, mouse and humans, respec-
tively. It contains the typical properties of G protein coupled
receptor (GPCR), namely, seven transmembrane domains of
20-25 amino acids predicted to form an -helice which spans
the plasma membrane and an extra cellular NH2 terminal
domain with glycosylation site. H1R is encoded by a single
exon gene that is located on the distal short arm of chromo-
some 3p25 in humans see in Fig. (2) and chromosome 6 in
mice. Histamine binds to aspartate residues in the transmem-
brane domain 3 of the H1-receptor, and to asparagine + ly-
sine residues within the transmembrane domain 5 [14].
Its structural studies done by photoaffinity binding prop-
erties using [125I]iodoazidophenpyramine (Table 1) and
subsequent sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE) analysis demonstrated that the H1-
receptor protein (molecular weight 56 kDa) is found under
reducing conditions in the brain of rat, guinea pig, and
mouse [79, 118, 129]. Similar studies have also been done
by using photoaffinity ligand [3H] azidobenzamide in bovine
adrenal medullar membranes and found labeled peptides in
the size range 53 to 58 kDa [130]. In guinea pig heart, the
specifically labeled H1R with [125I]iodoazidophenpyramine
was found to contain substantially higher molecular weight,
while there was no obvious difference in the characteristics
of the H1 R in tissues (Table 1) [131]. In 1991, H1R was
cloned from the bovine adrenal medulla by expression clon-
ing in the Xenopus oocyte system. Interestingly, 491 amino
acid protein with a calculated molecular weight of 56 kDa
was represented by the deduced amino acid sequence [130];
this protein has the seven transmembrane domains expected
of a G-protein coupled receptor (GPCR) and contains N-
terminal glycosylation sites. The main feature of the pro-
posed H1R structure is the very large 3rd intracellular loop
with 212 amino acids and relatively short intracellular C
terminal tail with 17 amino acids. The availability of the
bovine sequence and lack of introns has enabled the H1-
receptor to be cloned from several sp ecies including rat
[132], guinea pig [129, 133], mouse [134], and human [135,
136]. The human H1-receptor gene has now been localized
to chromosome 3 bands 3p14-p21 (Table 2). These clones
should be regarded as true species homologues of the H1-
receptor, while there are notable variations amongst them in
some antagonist potencies [23]. Nevertheless, it is clear that
the stereoisomers of chlorpheniramine show marked differ-
ences between species. For example, the guinea pig H1-
receptor has a KD of 0.9 nM for (1)-chlorpheniramine,
whereas for the rat H1-receptor, the value is nearly 8 nM
[23]. Similar variations for chlorpheniramine and other com-
pounds (mepyramine and triprolidine) have been shown in
guinea pig and rat brain, respectively [23, 67, 89]. On this
basis the species differences may explain why compound
[125I]iodobolpyramine can label guinea pig CNS H1-
receptors, but it is unable to iden tify H1Rs in the brain of rat
[78, 81]. In brain membranes of both guinea pig and rat the
native H1-receptor protein has been solubilized [137, 138],
and the solubilized receptor retains similar differences in H1-
antagonist potency for (1)-chlorpheniramine as that detected
in membranes [137]. It is important to note that mepyramine
seems to be potent antagonist of the recombinant rat H1-
receptor (i.e. expressed in C6 cells) than of the nativ e hista-
mine H1-receptor in the brain membrane of rat [23, 67, 132].
In addition, the recombinant stud ies performed in rat C6
cells [132] are complicated by the presence of a low level of
endogenous histamine H1-receptors (H1Rs) [139], but in the
functional studies in untransfected C6 cells, a high affinity
for mepyramine (KD 51 nM) has been deduced [23, 139].
The amino acid sequence alignment of the cloned histamine
H1- and H2-receptors led to the suggestion that the third and
fifth transmembrane domains (TM3 and TM5 resp ectively)
of receptor proteins are responsible for histamine binding
[140, 141]. In third transmembrane (TM3) of the human H1-
receptor, Aspartate (107) that is conserved in entire aminer-
gic receptors, has appeared to be essential for the histamine
binding, and also H1-receptor antagonists to the H1-receptor
[142]. In H1-receptor, the amino acid residues corresponding
to Asparagine (198) and Threonine (194) are in correspond-
ing positions in 5th transmembrane domain (TM5) of the
human H1-receptor, while the substitution of an Alanine for
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 15
Threonine (194) did not influence the binding properties of
either agonist or antagonist [142, 143]. However, the substi-
tution of Alanine (198) for Asparagine (198) decreased ago-
nist affinity, while the affinity of antagonist remained un-
CH
2
NCH
2
CH
2
N(CH
3
)
2
N
CH
3
O
Mepyramine
CHOCH
2
CH
2
N(CH
3
)
2
Diphenhydramine
N
CH
3
Br
CH
2
CH
2
CH
2
CH
2
NH N
N
CH
2
N
CH
3
O
H
Temelastine
S
N
CH2CHN(CH3)2
CH3
Promethazine
S
NCl
CH2CH2CH2N(CH3)2
Clorpromazine
CH
2
NCH
2
CH
2
N(CH
3
)
2
N
Tripelennamine
N
N
CH
2
NH NCH
2
CH
2
OCH
3
Astemizole
CCl N NCH2CH2OCH 2CO2H
H
Cetirizine
CNCH
2
CH
2
CH
2
CH
OH OH
C(CH
3
)
3
Terfenadine
N
N
Cl
H
Desloratadine
N
N
Cl
CO2CH2CH3
Loratadine
N
C=C
H
CH
2
N
CH
3
Triprolidine
HO
N
OH
O
OH
Fexofenadine
CHCH
2
CH
2
N(CH
3
)
2
N
Cl
Chlorpheniramine
Fig. (2). Chemical structures of some histamine H1-receptor-antagonists.
16 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
changed [142, 143]. Similar results have been seen in the
mutations to the corresponding residues Threonine (203) and
Asparagine (207) in the guinea pig-H1R sequence [144]. In
addition to these mutations 2-methylhistamine is affected by
the Asparagine (207) Alanine mutation, and H1-selective
agonists 2-thiazolylethylamine, 2-pyridylethylamine, and 2-
(3-bromophenyl) histamine are much less influenced through
this mutation [144, 145]. This suggested that Asparagine
(207) interacts with the Nt-nitrogen of histamine imidazole
ring.
However, it has been shown that Lysine (200) interacts
with the Np-nitrogen of histamine ring, and that it is impor-
tant for the activation of the H1R by histamine and the
nonimidazole agonist, 2-pyridylethylamine [63]. Further-
more, Leurs et al. [70] has demonstrated that the Lysine
(200) Alanine mutation did not alter the binding affinity of
2-pyridylethylamine to H1R of guinea pig. Thus, the studies
on the organization, genomic structure and promoter func-
tion of the human H1R revealed a 5.8 kb intron in the 50
flanking region of this gene, different binding sites for vari-
ous transcription factors, and the absence of TATA and
CAAT sequences at the appropriate locations [146].
3. Signaling Mechanisms
H1-receptor is a Gq/11-coupled protein with a very
large third intracellular loop and a relatively short C-terminal
tail see in Fig. (4). The main signal induced by ligand bind-
ing is the activation of phospholipase C-generating inositol
1, 4, 5-triphosphate and 1, 2-diacylglycerol (DAC) leading to
increased cytosolic Ca2+. The enhanced intracellular Ca2+
levels appear to account for the different pharmacological
properties promoted through the receptor including nitric
oxide (NO) production, liberation of arachidonic acid from
phospholipids, contraction of smooth muscles, dilatation of
arterioles and capillaries, vascular permeability in vessels as
well as stimulation of afferent neurons, and increased cAMP,
and also cGMP levels [64, 147] (see also Table 2). This re-
ceptor also stimulates nuclear factor kappa B (NFB) by
Gq/11 and G upon binding of agonist, while stimulation
of NFB occurs only via G leading to (pro)inflammatory
mediators [70, 89, 148]. The number of tissues and cell types
in which a H1R-mediated signals increases in either inositol
phosphate accumulation or intracellular calcium mobilization
has been described extensively and further details are pro-
vided in several comprehensive reviews [89, 149, 150]. In
Chinese hamster ovary (CHO) cells Ca2+ mobilization and
[3H]inositol phosphate accumulation has been observed due
to stimulation by histamine when CHO cells are transfected
with H1R-complementary deoxyribonucleic acid (cDNA) of
the human, bovine, and guinea pig [150, 151]. It is worth
demonstrating that in some tissues histamine can stimulate
inositol phospholipid hydrolysis independently of H1Rs.
Thus, in the longitudinal smooth muscle of guinea pig ileum
and neonatal rat brain [92, 152], a component can be identi-
fied in response to histamine that is resistant to inhibition by
H1R-antagonists. It is yet to be established whether these
effects are due to “tyramine-like” effects of histamine on
neurotransmitter release or direct effects of histamine on the
associated G-proteins [153, 154]. In addition to well known
effects on the inositol phospholipid signal transduction sys-
tems, several other signal transduction pathways can lead to
stimulation of H1R and it seems to be secondary to changes
in intracellular Ca2+ concentration or protein kinase C (PKC)
activation. Thus, nitric oxide synthase activity (via a
Ca2+/calmodulin-dependent pathway), and subsequent stimu-
lation of soluble guanylyl cyclase in a wide variety of vari-
ous cell types can be activated by histamine [155-158]. The
H1R can stimulate the arachidonic acid release and arachi-
donic acid metabolites synthesis such as prostacyclin and
thromboxane [150, 159]. It is being interestingly demon-
strated that the histamine-stimulated release of arachidonic
acid is partially inhibited (~ 40%) by pertussis toxin, when
CHO-K1 cells transfected with the guinea pig H1R and the
same response is also shown in HeLa cell possessing a native
H1R to resist pertussis toxin treatment [150]. The substantial
changes in the intracellular levels of cAMP can be produced
by H1-receptor activation, but in most tissues, H1R activa-
tion does not stimulate adenylyl cyclase directly, and acts for
the amplification of cAMP effects to histamine H2-, adeno-
sine A2-, and also vasoactive intestinal polypeptide receptors
[160-162]. The role of both intracellular Ca2+ ions and pro-
tein kinase C has been demonstrated in various cases in this
augmentation response [161]. H1R stimulation can also lead
to both cAMP responses and to an increasement of forskolin-
activated cAMP formation when CHO cells are transfected
with the bovine or guinea pig H1R [150, 163].
B. Histamine H2-Receptor
1. Cellular Distribution and Functional Characterization
The H2R is located on chromosome 5 in humans. Similar
to what has been demonstrated for H1R, the histamine binds
to transmembrane (TM) domains 3 (aspartate) and TM 5
(threonine and aspartate). The short 3rd intra-cellular loop
and the long C-terminal tail make a suitable feature of H2R
subtype, and the rat N-terminal extr acellular tail has N -
linked glycosylation sites [164]. Similar to H1R, H2R is ex-
pressed in different cell types (Table 2). It has been docu-
mented that H2R is mostly involved in suppressive activities
N
NN
CH
3
H
H
Methylhistaprodifen
N
NNH
2
F
3
C
H
2-[3-(Trifluoromethyl)phenyl]histamine
N
SNH2
2-(Thiazol-2-
yl)ethanamine
Fig. (3). Chemical structures of some histamine H1-receptor-agonists.
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 17
of histamine, while positive effects are mediated through
H1R. It was quite clear that the activation of H2R regulates
various functions of histamine including heart contraction,
gastric acid secretion, cell proliferation, differentiation and
immune response. It has been demonstrated that H2R an-
tagonists, such as zolantidine, is active in the treatment of
stomach and duodenal ulcers and it strongly suggests that the
clinical potency relates to the antagonistic effect of these
drugs on the secretion of stomach acids [14].
Hill [89] designed a study to map the distribution of
H2Rs by using radiolabeled H2R-antagonists, and achieved
more affinity with [3H] titotidine (Table 1) for the H2R in
guinea pig brain, lung parenchyma, and CHO-K1 cells trans-
fected with the human H2-receptor cDNA [165-167], but it
was not successful in the studies of rat brain [168]. The most
successful H2R-radioligand is [125I]iodoaminopotenti-dine,
which has high affinity (KD 50.3 nM) for the H2R in brain
membranes (Table 1) [129, 169-171] and also in CHO-K1
cells expressing the cloned rat H2R [171]. This compound
has also been used for autoradiographic mapping of H2Rs in
the brain of mammal [131, 170]. [125I]iodo-
aminopotentidine is also the most successful H2R-radio-
ligand (Table 1), which was used to map the distribution of
H2Rs in human brain with highest densities in the basal gan-
glia, hippocampus, amygdale, and cerebral cortex, and also
lowest densities were identified in cerebellum and hypo-
thalamus [170]. In guinea pig brain, a similar distribution has
been observed [129]. Irreversible labeling has also been suc-
cessfully seen by [125I]iodoazidopotentidine (Table 1) [129,
169]. H2R-stimulated cyclic AMP accumulation or adenylyl
cyclase activity in Fig. (4) has been shown in various tissues
including gastric cells, cardic tissue and brain [165, 172,
173] and gastric cells [174]. The potent effect of H2Rs have
been demonstrated on gastric acid secretion and the inhibi-
tion of this secretory process through H2R antagonists had
provided regulatory evidence for physiological role of his-
tamine in gastric acid secretion [175, 176]. In cardiac tissues
of most animal species, high concentrations of histamine
were present which can mediate positive chronotropic and
inotropic impacts on atrial or ventricular tissues by H2R
stimulation [177, 178]. Also H2R-mediated smooth muscle
relaxation has been documented in vascular smooth muscle,
uterine muscle and in airways [179-183]. Hill [89] had dem-
onstrated that the effects of H2Rs can inhibit a variety of
functions within the immune system. H2Rs have been shown
to negatively regulate the release of histamine on basophils
and mast cells [184, 185]. The inhibition of antibody synthe-
sis, T-cell proliferation, cell-mediated cytolysis, and cyto-
kine production were the increasing evidence of H2Rs on
lymphocytes [186-189]. The chemical structure of specific
H2R-antagonist and -agonists are shown in Figs. (5, 6).
2. Structural Biology of Receptor
The structural studies of H2R have been demonstrated
using [125I]iodoazidopotentidine and sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE) and it
was suggested that the H2R in guinea pig hippocampus and
striatum has a molecular weight of 59 kDa [129]. However,
comparison with the calculated molecular weights (40.2 to
40.5 kDa) for the cloned H2Rs indicates that the native H2R
in the brain of guinea pig was glycosylated. It was highly
significant with the proposal that entire cloned H2R proteins
Fig. (4). The classical binding sites of histamine and their main signaling pathways such as AC ( adenylate cyclase), PKC (protein kinase C),
PKA (protein kinase A), PLC (phospholipase C), H1+ or H2+ (stimulation via H1 or H2 receptor), H3- & H4- (inhibition via H3 and H4 re-
ceptors).
18 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
possess N-glycosylation sites in the N-terminus region [190-
192]. Fukushima et al. [193] has suggested that removal of
these glycosylation sites by site-directed mutagenesis
showed that N-glycosylation of the H2R is not essential for
cell surface localization, ligand binding, or coupling via Gs
to adenylyl cyclase. Gantz and colleagues for the first time
successfully cloned H2R using the polymerase chain reac-
tion to amplify a partial length H2R sequence from canine
gastric parietal cDNA using degenerate oligonucleotide
primers and this sequence was then used to identify a full
length H2R clone following screening of a canine genomic
library [167]. Following this cloning, many researchers have
cloned the rat, human, guinea pig, and mouse H2Rs [167,
191, 192, 194]. These intronless gene (DNA) sequences en-
code 359 amino acids for canine, human, guinea pig or 358
amino acids for rat receptor protein which has the general
properties of a G-protein-coupled receptor (GPCR) (Table
2). The radioligand binding studies using [125I]iodoamino-
potentidine were attempted to show the expression of rat and
human H2R proteins in CHO cells and revealed the expected
pharmacological specificity as shown in Table 1 [150, 171].
Chromosomal mapping studies have demonstrated that the
H2R gene was localized to human chromosome 5 [192].
Birdsall [140] has compared H2R sequence with other bio-
genic amine G-protein coupled receptors (GPCRs), and
demonstr ated th at an aspartate in transmembrane (TM) do-
main 3 and an aspartate and threonine residue in TM 5 were
more responsible for histamine binding. Replacement of as-
partate (98) with asparagine residue in the canine H2R pro-
vides significant results in a receptor that does not bind the
titotidine, an antagonist, and hence does not stimulate cyclic
adenosine monophosphate (cAMP) accumulation in hista-
mine response [195]. On changing the aspirate (186) residue
of TM 5 to an alanine residue there occurs complete loss of
the antagonist titotidine binding without affecting the EC 50
for cAMP formation in response to histamine stimulation.
Other change was observed on changing the threonine (190)
residue to an alanine residue, resulted in a lower KD for ti-
totidine antagonist and also a reduction in both the histamine
EC 50 value and maximal cAMP response [195]. Mutation
of Aspirate (186) and Glycine (187) residue in the canine
histamine H2-receptor to Alanine (186) and Serine (187)
residue produces a bifunctional receptor, which can be acti-
vated through adrenaline, and inhibited via both cimetidine
and propranolol [196]. Thus, these results indicate that
pharmacological specificity of the H2R resides in only lim-
ited key amino acid residues.
3. Signaling Mechanisms
H2R is coupled both to adenylate cyclase and to phos-
phoinositide second messenger systems via separate GTP-
dependent mechanisms. Receptor binding stimulates activa-
tion of c-Fos, c-Jun, protein kinase C (PKC) and
p70S6kinase [14, 89, 145, 174] see in Fig. (4). Histamine
N
N
S
NN
CH
3
CH
3
N
NC
HH
H
Cimetidine
HN N
NNCH
3
CH
3
H
Mifentidine
HC NO
2
SN
N
H
3
C
CH
3
S
HN
NCH
3
H
Nizatidine
HC NO2
O
N
H3C
CH3
S
HN
NCH
3
H
Ranitidine
NNH
2
H
2
NNS
S
NN
CH
3
N
NC
HH
Titotidine
N
SNH
2
O
O
H
2
N
NH
2
SN
N
S
NH
2
Famotidine
N
ON
S
N
H
Zolantid ine
N
ON
N
CN
NN
O
I
NH
2
H
H
H
Iodoaminopotentidine
Fig. (5). Chemical structures of some histamine H2-receptor-antagonists.
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 19
was proved to be a highly potent stimulant of cAMP accu-
mulation in various cells, and H2R-dependent impacts of
histamine were predominantly mediated through cAMP [14,
174], particularly those of central nervous system (CNS)
origin [23]. Thus, H2R-mediated impacts on cAMP accumu-
lation have been well documented and had been demon-
strated in brain slices, gastric mucosa, fat cells, cardiac myo-
cytes, vascular smooth muscle, basophils and neutrophils
[23, 172, 197-199]. In addition, H2R-mediated cAMP accu-
mulation had been observed in Chinese hamster ovary
(CHO) cells transfected with the rat, canine, or human H2R
cDNA [167, 190, 193, 200]. In both brain and cardiac mus-
cle membranes, the direct stimulation of adenylyl cyclase
activity in cell free preparations had been detected [201,
202].
However, Hill [89] had suggested that the caution is re-
quired regarding the interpretation of receptor characteriza-
tion studies using histamine-stimulated adenylyl cyclase ac-
tivity alone. A most striking feature of studies of H2R-
stimulated adenylyl cyclase activity in membrane prepara-
tions was the potent antagonism demonstrated with certain
neuroleptics and antidepressants [204]. In intact cellular sys-
tems, most of the neuroleptics and antidepressants were ap-
proximately 2 orders of magnitude weaker as antagonists of
histamine-stimulated cAMP accumulation [203, 205]. One
highly potential explanation of these variations resides
within the buffer systems used for the cell-free adenylyl cy-
clase assays, and some differences in potency of some anti-
depressants and neuroleptics have been demonstrated when
membrane binding of H2Rs has been evaluated using
[125I]iodoaminopotentidine (Table 1). However, the varia-
tions observed in the Ki values deduced from studies of
ligand binding in different buffers are not as large as the
variations in KB values obtained from functional studies. For
example, in the case of amitriptyline, no difference was ob-
served in binding affinity in Krebs and Tris buffers [206]. In
addition to Gs-coupling to adenylyl cyclase, H2Rs are cou-
pled to other signaling systems also. For example, H2R
stimulation has been demonstrated to enhance the intracellu-
lar free concentration of calcium (Ca2+) ions in gastric parie-
tal cells [207, 208]. In some cell systems, Gq coupling to
PLC and intracellular Ca2+ had been demonstrated (Table 2).
In HL-60 cells, a similar calcium (Ca2+) response to H2R
stimulation had been demonstrated [209], and similar case
was observed in hepatoma-derived cells transfected with the
canine H2Rs cDNA [210]. Therefore, the influence on
[Ca2+]i was accompanied by both an increase in inositol
trisphosphate accumulation and a stimulation of cAMP ac-
cumulation in these latter cells [210]. It was interesting to
note that in these cells the H2R-stimulated calcium and
inositol trisphosphate responses were both inhibited by chol-
era toxin treatment, whereas cholera toxin produced the ex-
pected increase in cAMP levels [208, 210]. H2Rs release
Ca2+ from intracellular calcium stores in single parietal cells
[211] and no effect of H2R agonists was observed on intra-
cellular calcium levels or inositol phosphate accumulation in
CHO cells transfected with the H2R of human [144]. Thus,
the effect of H2R stimulation on intracellular Ca2+ signaling
may be highly cell-specific.
The stimulation of H2R produces both inhibition of P2u-
receptor-mediated arachidonic acid release and an increase in
cAMP accumulation in CHO cells transfected with the rat
H2R [171]. However, Traiffort et al. [171] had demonstrated
that the effect on phospholipase A2 activity (i.e., arachidonic
acid release) was not mimicked by forskolin, PGE1, or 8-
bromo-cAMP, suggesting a mechanism of activation that is
independent of cAMP-mediated protein kinase A activity.
However, inhibitory effects of H2R stimulation were ob-
served on phospholipase A2 activity in CHO cells trans-
fected with the human H2R [200]. Thus, these cAMP-
independent effects might depend on the level of receptor
expression or subtle differences between clonal cell lines.
H
H
HN N
NN
NH
N
F
Arpromidine
NS
NH
2
CH
3
NH
2
Amthamine
HN N
NN
S
NH
N
N
CH
3
HH
H
Impromidine
S
H
2
NNH
N
CH
3
CH
3
Dimaprit
HN N
NN
S
CH
3
NH NHN
CH
3
HH
Sopromidine (*: R chirality)
NNH
NH
2
CH
3
4-Methylhistamine
Fig. (6). Chemical structures of some histamine H2-receptor-agonists.
20 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
C. Histamine H3-Receptor
1. Cellular Distribution and Functional Characterization
The function of histamine as neurotransmitter has been
proved with the discovery of the H3R. It was mainly in-
volved in brain functions, also the peripheral effect of hista-
mine on mast cells via H3Rs, which mainly involves the
nervous system, and might be connected to a local neuron-
mast cell interaction [212]. Its involvement in cognition,
sleep-wake status, energy homeostatic regulation and in-
flammation had attracted many pharmaceutical researches
for numerous, so far unmet, therapeutic approaches in d iffer-
ent peripheral, but mainly central diseases [213, 214]. A re-
cent study had reported that it is presynaptically located as
autoreceptor controlling the synthesis and release of hista-
mine [215]. It was observed that H3-autoreceptor activation
stimulates the negative feedback mechanism that reduces
central histaminergic activity [216]. H3R’s heterogeneity in
binding and its functional studies has well been documented,
and suggested more than one H3R subtype. This assumption
had been confirmed by demonstration of several H3R vari-
ants, generated from the complex H3R gene by alternative
splicing. The three functional isoforms have been found in
the rat, and they all vary in length of the 3rd intracellular
loop, their distinct central nervous system (CNS) localiza-
tion, and differential coupling to adenylate cyclase and
MAPK signaling. Similar results in case of humans were
obtained [217-219].
Thus, numerous isoforms are found in different species
and different tissues leading to the assumption that signaling
fine-tuning may be controlled via receptor oligomerization
or formation of isoforms [220].
H3R is anatomically localized primarily to the CNS with
prominent expression in basal ganglia, cortex hippocampus
and striatal area. In the periphery, H3R can be found with
low density in gastrointestinal, bronchial and cardiovascular
system [221]. The high apparent affinity of R-()-
methylhistamin e for the H3R has enabled the use of this
compound as a radiolabeled probe (Table 1) [222]. In rat
cerebral cortical membranes, this compound (R-()-
methylhistamine) has been used to identify a single binding
site, and which in phosphate buffer has the important phar-
macological characteristics of the H3R [222, 223]. In rat
brain membranes, [3H]R-()-methylhistamine binds with
high affinity (KD 50.3 nM), although its binding capacity is
low (~ 30 fmol/mg protein) [222]. It was significantly nota-
ble that the autoradiographic studies with [3H]R-()-
methylhistamine have described the presence of specific
thioperamide-inhibitable binding in several rat brain regions,
especially cerebral cortex, striatum, hippocampus, olfactory
nucleus, and the bed nuclei of the stria terminalis, which
receive ascending histaminergic projections from the mag-
nocellular nuclei of the posterior hypothalamus [222, 224].
In human brain and the brain of nonhuman primates, the
H3Rs have also been visualized [225]. H3R binding has also
been characterized using [3H]R-()-methylhistamine in
guinea pig lung [222], guinea pig cerebral cortical mem-
branes [226], guinea pig intestine and guinea pig pancreas
[227]. N-methylhistamine as a radiolabeled probe had
proved successful for the H3R (Table 1). The relative agonist
activity of N-methylhistamine (with respect to histamine)
was significantly similar for all three histamine receptor
(HRs) subtypes, but the binding affinity of histamine and
N-methylhistamine for the H3R was several orders of mag-
nitude higher than for either H1- or H2-receptors [23, 131].
N-methylhistamine can identify high-affinity H3R sites in
both rat [228, 230] and guinea pig [227] brain. The binding
of H3-receptor-agonists to H3Rs in brain tissues was found
to be regulated by guanine nucleotides, implying its relation
to heterotrimeric G-proteins [222, 223, 228, 229]. Also the
binding of H3R agonists appears to be more sensitive to sev-
eral cations. For example magnesium (Mg2+) and sodium
(Na+) ions inhibit [3H]R-()-methylhistamine binding in
guinea pig and rat brain [226], and the presence of calcium
(Ca2+) ions has been shown to reveal heterogeneity of ago-
nist binding [223]. It is important to note that the inhibitory
effect of sodium (Na2+) ions on agonist binding means higher
Bmax values that were usually obtained in sodium-free Tris
buffers compared with the Na/K phosphate buffers [228].
The multiple histamine H3R subtypes exist in rat brain
(termed H3A and H3B) on the basis of [3H]N-
methylhistamine binding in rat cerebral cortical membranes
in 50 mM Tris buffer (Table 1) [230]. Based on these condi-
tions, the selective histamine H3-antagonist thioperamide
can discriminate two affinity-binding states [230]. However,
Heterogeneity of thioperamide binding was sodium (Na2+)
ions concentration dependent or depends on guanine nucleo-
tides within the incubation medium [228]. Thus, in the pres-
ence of 100 mM sodium chloride, thioperamide binding con-
forms to a single binding isotherm [228], and H3R can exist
in different conformations for which thioperamide, but not
agonists or other H3R-antagonists (clobenpropit) can dis-
criminate. This suggests that the equilibrium between these
conformations is altered by guanine nucleotides or sodium
(Na2+) ions [228]. If this speculation is correct, it is likely
that the different binding sites represented resting, active, or
G-protein-coupled conformations of the H3R. Furthermore,
if thioperamide preferentially binds to uncoupled receptors,
then this compound should exhibit negative efficacy in func-
tional assays. Radiolabeled H3R antagonists [125I]iodo-
phenpropit, has been used to label histamine H3Rs in rat
brain membranes (Table 1) [231]. The inhibition curves for
iodophenpropit and thioperamide were consistent with inter-
action with a single binding site, but H3R agonists were
found to be able to discriminate both high- [4 nM for R-()-
methylhistamine] and low- [0.2 mM for R-()-methyl-
histamine] affinity binding sites [231]. [3H]GR16820 and
[125I]iodoproxyfan have been proved useful as high-affinity
radiolabeled H3R-antagonists [232, 233]. In rat striatum, in
the IUPHAR classification of histamine receptors 267 pres-
ence of guanine nucleotides such as guanosine 59O- (3-
thiotriphosphate) (GTPgS), 40% of the binding sites exhib-
ited a 40-fold lower affinity for H3-agonists, providing fur-
ther evidence for a potential linkage of H3Rs to G-proteins.
In rat brain membranes, [3H]thioperamide and [3H]5-
methylthioperamide, both have been used to label H3R
[234]. However, [3H]thioperamide was shown to bind addi-
tionally to low affinity, high-capacity, non H3R sites [234].
The localization of H3Rs had come out from functional stud-
ies, primarily involving inhibition of neurotransmitter re-
lease. The H3R was first characterized as an auto receptor
regulating histamine synthesis and release from rat cerebral
hippocampus, cortex, and striatum [222, 235]. In human
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 21
cerebral cortex, the H3R-mediated inhibition of histamine
release has also been demonstrated [235]. Differences in the
distribution of H3R binding sites and the levels of histidine
decarboxylase (an index of histaminergic nerve terminals)
suggested at an early stage that H3Rs were not confined to
histamine-containing neurons within the mammalian CNS
[222, 236]. It has been documented by the observations that
H3Rs can regulate neurotransmitter release in mammalian
brain as serotonergic, noradrenergic, cholinergic, and dopa-
minergic [237-240]. H3R activation inhibits the firing of the
histamine-neurons in the posterior hypothalamus by a
mechanism different from auto-receptor functions found on
other aminergic nuclei, and presumably a block of Ca2+ cur-
rent [241]. H3Rs were found to regulate the release of sym-
pathetic neurotransmitters in guinea pig mesenteric artery
[242], human saphenous vein [243], guinea pig atria [244,
245], and human heart [246].
An important inhibitory effect of H3R activation on re-
lease of neuropeptides (tachykinins or calcitonin gene-
related peptide) from sensory C fibers has been investigated
from airways [247], meninges [248], skin [249], and heart
[250]. The modulation of acetylcholine, capsaicin, and sub-
stance P effects by H3Rs in isolated perfused rabbit lungs
has also been reported [251]. There was evidence that H3R
activation can inhibit the release of neurotransmitters from
nonadrenergic- noncholinergic nerves in guinea pig bronchi-
oles [252] and ileum [253]. In guinea pig ileum, the H3R-
antagonists betahistine and phenylbutanoylhistamine were
much less potent as inhibitors of H3R-mediated effects on
nonadrenergic-noncholinergic transmission than they were
as antagonists of histamine release in rat cerebral cortex
[253].
A similar low potency has been investigated for betahis-
tine and phenylbutanoyl histamine antagonists for antago-
nism of H3R-mediated [3H]acetylcholine release from rat
entorhinal cortex [239], and antagonism of H3R-mediated 5-
hydroxytryptamine release from porcine enterochromaffin
cells [254]. These investigations provide possible support for
the existence of distinct H3R subtypes and it had been shown
that phenylbutanoylhistamine can inhibit [3H] acetylcholine
release from rat entorhinal cortex slices, and synaptosomes
by a nonhistamine receptor mechanism [255]. Therefore, the
potency of phenylbutanoylhistamine as H3R-antagonist in
those preparations can be highly underestimated because of
the additional nonspecific activities of the drug [255]. The
inhibitory effect of H3-receptor stimulation on 5-HT release
from porcine enterochromaffin cells in strips of small intes-
tine [254] provides evidence for H3-receptors regulating
secretory mechanisms in non-neuronal cells. Hence, it can be
concluded that H3R may be present in gastric mast cells or
enterochromaffin cells and exert an inhibitory effect on his-
tamine release and gastric acid secretion. In conscious dogs,
H3R activation had been observed to inhibit gastric acid se-
cretion [256], and in isolated rabbit fundic mucosal cells. An
autoregulation of histamine synthesis by H3R had also been
investigated [257]. It had been demonstrated that H3R re-
laxes rabbit middle cerebral artery by an endothelium-
dependent pathway involving both nitric oxide and prosta-
noid release [258, 259]. H3-receptor stimulation can activate
adrenocorticotropic hormone release from the pituitary cell
line AtT-20 [260]. Therefore, H3R provides constitutive
properties, which means part of the receptor population
spontaneously undergoes allosteric transition leading to a
conformation, to which G protein can bind [261, 262], and
also H3R-knock out mice manifest an obese phenotype
(characterized through increased body weight, food intake,
adiposity, and reduced energy expenditure). Recently, it has
been observed that H3R express insulin and leptin resistance
as well as a diminution of the energy homeostasis-associated
genes UCP1 and UCP3 [263]. The chemical structure of spe-
cific H3R-antagonists and –agonists are shown in Figs. (7,
8).
2. Structural Biology of Receptor
The H3-receptor is also G protein-coupled (GPCR) and
had been cloned [22]. Its gene consists of 4 exons spanning
5.5 kb on chromosome 20 (20q13.33) in humans (Table 2).
Structural studies of H3R are very limited and there are only
few reports on its purification studies. By using
[3H]histamine as a radioligand, the solubilization of a H3R
protein from bovine whole brain has been reported and then
Size-exclusion chromatography has revealed an apparent
molecular mass of 220 kDa [229]. However, because the
solubilized receptor retained its guanine nucleotide sensitiv-
ity and it is likely that the molecular mass of 220 kDa repre-
sents a complex of receptor, G-protein, and digitonin [229].
Cherifi et al. [264] have reported the solubilization (with
Triton X-100) and purification of the H3-receptor protein
from the human gastric tumoral cell line HGT-1. After gel
filtration and sepharose-thioperamide affinity chromatogra-
phy, protein has been purified with a molecular mass of ap-
proximately 70 kDa (see Table 2).
3. Signaling Mechanisms
The signal mechanisms used by the H3R remain largely
subject to hypothesis, but there is increasing evidence to
suggest that this receptor belongs to the G-protein-coupled
receptors (Gi/o) (Table 2), and its activation leads to inhibi-
tion of cAMP formation, accumulation of Ca2+ and stimula-
tion of mitogen-activated protein kinase (MAPK) pathway
[212], see Fig. (4). This evidence had been obtained from
ligand-binding studies that involve the modulation by gua-
nine nucleotides of H3R-agonist binding [223, 226, 228-230]
and H3R-agonist inhibition of H3R-antagonist binding [231,
233, 265]. The direct evidence for a functional H3R-G-
protein linkage came from studies of [35S]GTPgS binding to
rat cerebral cortical membranes [266]. In rat cerebral cortical
membranes, the presence of H1R- and H2R-antagonists (0.1
mM mepyramine and 10 mM titotidine), and both R-()-
methylhistamine and N-()-methylhistamine generated a
concentration dependent stimulation of [35S]GTPgS binding
(EC50 = 0.4 and 0.2 nM) [266]. Notably, this response was
inhibited via pretreatment of membranes with pertussis
toxin, and implying a direct coupling to a Gi or Go protein
[266]. The evidence of pertussis toxin-sensitive G-proteins in
the response to H3R stimulation came from studies of H3R
signaling in human and guinea pig heart [244, 246]. H3R-
activation appeared to lead to an inhibition of N-type Ca2+
channels responsible for voltage dependent release of
noradrenaline in human and guinea pig heart [244, 246], but
several investigations have failed to demonstrate an inhibi-
tion of adenylyl cyclase activity in different tissues and cells
[264, 267] which might suggest that H3Rs couple to Go pro-
teins.
22 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
D. Histamine H4-Receptor
1. Cellular Distribution and Functional Characterization
The discovery of the H4-receptor adds a new chapter to
the histamine story. The H4 R is preferentially expressed in
intestinal tissue, spleen, thymus, medullary cells, bone mar-
row and peripheral hematopoietic cells, including eosino-
phils, basophils, mast cells, T lymphocytes, leukocytes and
dendritic cells. However, moderate positive signals have also
been detected in brain, spleen, thymus, small intestine, co-
lon, heart, liver and lung. Although first expression studies
demonstrated the absence of H4Rs in the central nervous
system (CNS), but in situ hybridization studies suggested
evidence for their human brain localization in low density.
The relatively restricted expression of the H4R provides an
important role in inflammation, hematopoiesis and immunity
by the regulation of H4R expression via stimuli such as IFN,
TNF- and IL-6, IL-10, and IL-13. Basophils and mast cells
express H4R-mRNA . The H4R mediates chemotaxis of mast
cells and eosinophils as well as control cytokine release from
dendritic cells and T cells. It was demonstrated that the H4R
is participated, along with the H2R, in the control of IL-16
release from human lymphocytes. It had also been hypothe-
sized that H4R selective antagonist might be useful in help-
ing to treat anti-inflammatory potency in models of asthma,
arthritis, colitis and pruritis. Antagonists, such as JNJ
7777120, have also been shown to be effective in various
model of inflammation. Up to now, very little is known
about the biological functions of H4R. There are few reports
in the literature, providing evidence for chemotactic activity
in mast cells and eosinophils or control of IL-16 production
by CD8+ lymphocytes. It suggests an important role of H4R
in the regulation of immune function and offers novel thera-
peutic potentials for histamine receptor ligands in allergic
and inflammatory diseases [268-280]. A recent study showed
the role of H4R in mast cell, eosinophil, and T cell function,
as well as the effects of its antagonist, JNJ 7777120, in a
mouse peritonitis model pointing to a more general role for
H4R in inflammation. Selective H4R antagonists like JNJ
7777120 shows potential role in treatment of inflammation
in humans. In many diseases such as allergic rhinitis, asthma,
and rheumatoid arthritis, conditions where eosinophils and
mast cells are involved, H4R antagonists have therapeutic
utility [281]. The discovery of H4R and its emerging role in
inflammation had spurred new interest for the functions of
histamine in inflammation, allergy and autoimmune diseases.
Early results in animal models suggest that H4R antagonists
may have utility in treating various conditions in humans, in
particular, in diseases in which histamine is known to be
present and in which H1R antagonists are not clinically ef-
fective [282]. Obviously, a better functional characterization
of H4R benefits from the exploitation of new, specific tools,
such as the recently develop ed potent and selective non-
imidazol H4R antagonist [281]. It can be expected that the
role of H4R will be more important in autoimmune disor-
ders, allergic conditions and nociceptive responses in the
N
NN
S
HN
H
Thioperamide
H
N
HN
SN
NH
Cl
Clobenpropit
H
N
HN
SN
NH
I
Iodophenpropit
N
HN
NH
2
Impentamine
N
HN
N
N
O
CH
2
Cl
GR 174737
N
HN
NN
SN
HN
CH
3
NH
H
H
Im-
promidine
Fig. (7). Chemical structures of some histamine H3-receptor-antagonists.
HN N
SNH
NH2
Imetit
HN N NH
Immepip
HN N
C
NH2
H
CH3
(R)--Methylhistamine
Fig. (8). Chemical structures of some histamine H3-receptor-agonists.
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 23
near future. The chemical structure of specific H4R-
antagonists and –agonists are shown in Fig. (9).
2. Structural biology of Receptor
The human H4-receptor gene was mapped to chromo-
some 18q11.2 which encodes a 390 amino acid and also re-
lated to seven transmembrane G-protein coupled receptor. It
shares 37-43% homology (58% in transmembrane regions)
with the H3-receptor and a similar genomic structure. The
H4R gene spans more than 21 kbp and contains three exons,
separated by two large introns (>7 kb) (Table 1) with large
interspecies variations from 65-72% homology in sequences.
Analysis of the 5 flanking region did not reveal the canoni-
cal TATA or CAAT-boxes. The promoter region contains
several putative regulatory elements involved in proinflam-
matory cytokine signaling pathways. H4Rs are coupled to
Gi/o, which initiates various transduction pathways such as
inhibition of forskolin-induced cAMP formation, enhanced
calcium influx and MAPK activation. In accordance with th e
homology between the two receptors, several H3R-agonists
and antagonists were recognized by the H4R, although with
different affinities. It has been observed that H3R-agonist R-
-methyl histamine acts on H4R with several hundred times
less poten cy. Similar effect has been seen with thioperamide,
the classical H3R antagonist which also behave like a H4R
antagonist (Table 1), though with a much lower affinity and
clobenpropit, also a H3R antagonist, which exerts agonistic
activity on H4R [Table 1; 21, 273, 274, 283-286].
Albeit histamine binding to H4R is very similar to that
reported for the other histamine receptors (it shows the im-
portance of the Asp 94 residue in transmembrane region
(TM) 3 and the Glu 182 residue in the TM 5) however, some
differences exist and these were exploited to design specific
tools. Mouse, rat and guinea pig H4Rs have been cloned and
characterized and were found to be only 68, 69, and 65%
homologous respectively to their human counterpart. These
studies have revealed substantial pharmacological variations
between species, with higher affinity of histamine for human
and guinea pig receptors than for their rat and mouse equiva-
lents [287].
3. Signaling Mechanisms
The signal mechanisms used by the H4R remain highly
subject to the G-protein-coupled receptors (Gi/o), and its
activation leads to an inhibition of adenylyl cyclase and
downstream of cAMP responsive elements (CRE) as well as
activation of mitogen-activated protein kinase (MAPK) and
phospholipase C with Ca2+ mobilization (Table 2); see Fig.
(4).
IV. HISTAMINE: NON-CLASSICAL BINDING SITES
A. Cytochrome P450
The human cytochrome P450 (CYP450) superfamily
comprises 57 genes encoding heme-containing enzymes,
which are found in the liver as well as in extrahepatic tissues
(adrenals, and peripheral blood leukocytes), where they can
be stimulated by various stimuli [288, 289] (Fig. 10). They
are not only involved in metabolism of large number of for-
eign substances, but also play an important role in diverse
physiological processes [generation, transformation or inac-
H4-receptor antagonists
N
N
N
Cl
O
H
JNJ 7777120
N
N
N
N
Cl
O
H
JNJ 10191584
N
NN
S
HN
H
Thioperamide (partial H4R-antagonist)
H4-receptor agonists
N
NNH2
H
4-Methylhistamine
H
N
HN
SN
NH
Cl
Clobenpropit (partial H4R-agonist)
Fig. (9). Chemical structures of specific H4-receptor-antagonists and -agonists.
24 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
tivation of endogenous ligands (steroids and lipids)], which
are involved in cell regulation [290].
Binding of histamine to CYP450 had been shown by
Branders, who proposed a second messenger role for intra-
cellular histamine via this binding site. This hypothesis was
mainly based on a finding that N, N diethyl-2-(4-
(phenylmethyl)phenoxy) ethanamine (DPPE), an arylalky-
lamine analogue of tamoxifen inhibits the binding of hista-
mine to CYP450 [291]. DPPE allosterically modify hista-
mine binding to the heme moiety of CYP450 enzymes and
inhibited platelet aggregation, as well as lymphocyte and
hematopoietic progenitor proliferation [292, 293]. The effect
of DPPE on histamine binding was found to be highly com-
plicated and depends on the nature of the P450 enzymes.
Thus, it inhibits the action of histamine on CYP2D6 and
CYP1A1, which enhances its effect on CYP3A4 and does
not affect CYP2B6 [294]. The heme moiety of CYP450
binds to several histamine antagonists [295, 296], particu-
larly H3R antagonists (thioperamide, clobenproprit and
ciproxyfan) [297]. This property explains some effects of
these antagonists, when used at high doses. Notably, hista-
mine interacts with CYP450 and it has been demonstrated
that CYP2E1 and CYP3A were upregulated in histidine de-
carboxylase (HDC)-deficient mice [298].
B. Transporters of Histamine
Histamine (2-(1H-imidazol-4-yl) ethanamine) is synthe-
sized in the cytosol and requires a specific transport into se-
cretory vesicules where it is sequestered. Vesicular mono-
amine transporters (VMATs) are proteins, which accomplish
this specific task for several neurotransmitters [299] (Fig.
10). The two subtypes of monoamine transporters are
VMAT1 and VMAT2 (that have been cloned and character-
ized) but VMAT2 can transport histamine. Vesicular mono-
amine transporter 2 (VMAT2) had been cloned from rat and
human brain, bovine adrenal medulla and a basophilic leu-
kemia cell line. When histamine biosynthesis was enhanced
then its expression was found to be up regulated by several
stimuli. The increased VMAT2 expression in IL-3-
dependent cell lines was enhanced with enhanced histamine
synthesis in response to calcium (Ca2+)ionophore [300].
VMAT2 is responsible for the transport of histamine into
secretory granules of enterochromaffin-like (ECL) cells. The
gene expression of VMAT2 was found to be modulated via
cytokines, either positively (TGFa) or negatively (IL-1 and
TNF-) [301]. VMAT2-deleted granules do not release his-
tamine upon activation, even though granule cell fusion does
still occur [302]. The bone marrow-derived mast cells from
histidine decarboxylase (HDC)-deleted mice are completely
devoid of endogenous histamine but can take up the media-
tor from histamine-supplemented medium and store it in
secretory granules. Hence, two transporters are essential to:
1. insure the passage across the plasma membrane, and
2. cross the vesicular membrane.
First transporter has not been identified yet, but the sec-
ond transporter seems to be vesicular monoamine transporter
2 (VMAT2). The non-neuronal monoamine transporters that
actively remove monoamines from extracellular space have
been described as organic cation transporter 1 (OCT1),
OCT2, and extraneuronal monoamine transporter (EMT).
EMT was also designated as OCT3. The expression of OCT1
was found to be restricted to liver, kidney and intestine,
OCT2 in brain and kidney, while EMT showed a broad tis-
sue distribution. It has been established that OCT1 cannot
transport histamine, conversely to OCT2 and EMT for which
Fig. (10). The non-classical histamine binding sites and their main signaling pathways such as DAO: diamine oxidase; HMT: histamine
methyl transferase; OCT: organic cation transporter; HDC: histidine decarboxylase; CYP 450: cytochrome P450; VMAT: vesicular mono-
amine transporter.
Histamine Receptors in Immunomodulation The Open Immunology Journal, 2009, Volume 2 25
it is a good substrate [303]. Thus, EMT appeared to be a
good candidate as histamine transporter in mast cells and
basophils, accounting for their capacity to take up the media-
tor from the environment.
V. IMMUNE REGULATION BY HISTAMINE IN IM-
MUNOMODULATION AND ALLERGIC INFLAM-
MATION
Histamine exerts a very important immunomodulatory
effect via H1-, H2-, H3-, and H4-receptors [1, 20, 128, 188,
189, 270, 304; Table 3]. According to the cell differentiation
stage and microenvironment influences, the receptors ex-
pression changes. Histamine shows proinflammatory or anti-
inflammatory effects, depending on the predominance of the
type of histamine receptor (H1R, H2R, H3R & H4R) and on
the experimental system studied. Histamine had proinflam-
matory activity through the H1R, and is involved in the de-
velopment of various aspects of antigen-specific immune
response including the maturation of dendritic cells (DCs)
and the modulation of the balance of type 1 helper (Th1) T
cells and type 2 helper (Th2) T cells. Histamine blocks hu-
moral immune responses by means of a specific mechanism
in which it induce an increase in the proliferation of Th1
cells and in the production of interferon (IFN- ). Hista-
mine also stimulates the release of proinflammatory cytoki-
nes and lysosomal enzymes from human macrophages and
shows the capacity to influence the activity of immune cells
including mast cells, basophils, eosinophils, fibroblasts,
lymphocytes, neutrophils, epithelial and endothelial cells.
The role of histamine in auto immunity and malign ant disease
through the H1R is well documented [20, 128]. Histamine
also plays a pivotal role in allergic inflammation which is a
complex network of cellular events and involves redundant
mediators and signals. Histamine is released from the gran -
ules of mast cells and basophils (FcRI+ cells) along with
several mediators such as tryptase, leukotrienes, prostagland-
ins, and other newly generated mediators. Histamine was
found in relatively large (g) quantities per 1 million cells, in
contrast to leukotrienes and other mediators (which are pre-
sent in picograms), after allergen challenge in sensitized per-
sons. Most of the potent effects of histamine in allergic in-
flammation occur through H1Rs [1, 13, 20; Table 3], while
hypotension, flushing, headache, and tachycardia occur both
by the H1- and H2-receptors in th e vasculature [305].
Whereas, nasal congestion and cutaneous itch occurs both by
the H1- and H3- receptors [306, 307]. Histamine also acts as
a contributor to the late allergic response by generating a
stimulatory signal for the production of cytokines, the ex-
pression of cell adhesion molecules and class II antigens.
VI. EFFECT OF HISTAMINE IN IMMUNE CELLS
WITH RESPECT TO ALLERGIC DISEASES
Histamine’s classical effects, expressed at the organ
level, have been documented and were highly emphasized in
allergies and autoimmune diseases. Histamine directly or
indirectly influences the activity of various inflamma-
tory/effector/immunologic cell types involved in the patho-
genesis of several diseases. Indeed, several studies have sug-
gested that histamine receptors (HRs) are expressed on mast
cell and basophils; lymphocytes; neutrophils; monocytes,
macrophages and dendritic cells (DCs); eosinophils; epithe-
lial cells; endothelial cells, and therefore modulate the func-
tion of these cells in immune system.
A. Mast Cells and Basophils
Recent studies shed light on the potent role of histamine
in mast cells and basophils, both types of cells can them-
selves be modulated by histamine as they express H1-, H2-
and H4-receptors [271, 308, 309]. The peritoneal and skin
mast cells exhibited aberrant granules with very low electron
density, in HDC-deficient mice, which indicated the drastic
decrease in the granule contents including granule proteases
and sulfated proteoglycans [310]. The critical roles of hista-
mine in cutaneous and systemic anaphylaxis have been sug-
gested by using the HDC-deleted mice [311, 312] and it re-
mained a possib ility that diminished granule constituents,
such as proteases, make contribution to the relief of anaphy-
laxis in the mutant mice. How h istamine regulates allergic
responses by maturation of tissue mast cells requires to com-
prehend detailed studies on the effect of absence of hista-
mine on mast cell function. Impact of histamine was also
demonstrated in the migration of mast cells which was medi-
ated exclusively through the H4R [271]. It has been shown
that histamine acting through H4Rs can stimulate chemo-
taxis of murine mast cells in vitro [271] and lead to changes
in tissue localization in vivo [281]. A hematopoietic organ,
bone marrow, contains certain types of cells which can pro-
duce histamine in response to IL-3 [30, 313]. The role of IL-
3 -sensitized histamine synthesis in bone marrow remains to
be clarified [39], however, a study suggested a unique circuit
of newly synthesized histamine and its implication in baso-
phil precursors [314]. It has been documented that bidirec-
tional transport of histamine is facilitated largely through
organic cation transporter 3 (OCT3) in the plasma mem-
branes of the FcRI+, c-kit bone marrow cells. It had been
demonstrated that intracellularly stimulated h istamine in the
organic cation transporter 3 (OCT3)-deleted cells has sup-
pressive impacts on expression of HDC, IL-4, and IL-6. This
suggests not only the feedback inhibition of histamine syn-
thesis but also the suppression of Th2 cytokine production
through immature basophils [303, 315]. In addition, hista-
mine receptor binding studies with specific receptor antago-
nists have suggested that basophils express predominantly
H2R, and these were involved in the regulation of IgE-
stimulated histamine release, as demonstrated through in-
creased histamine release in the presence of anti-IgE and
cimetidine (a H2R antagonist) but not in the presence of anti-
IgE and thioperamide (a H3R antagonist) [316-318]. H2Rs
in mast cells show various effects such as inhibition of his-
tamine release and modulation of cytokine production [319].
It has been suggested that H3R functions on mast cells but
many of these properties may be attributed to the H4R as the
ligands used were not specifically selective. H3R expressio n
was not detected in some types of mast cells [271].
B. Lymphocytes
The expression of histamine receptors (HRs) on the cell
surface of immunocompetent cells, including lymphocytes
(B-cells and T-cells) and their effects mediated by receptors
(HRs) have been published in several studies and signifi-
cantly reviewed [320] (Fig. 11). It has been concluded that
both histamine receptors (H1 and H2) are present on the
26 The Open Immunology Journal, 2009, Volume 2 Shahid et al.
lymphocytes but there is only few data available on the func-
tional significance of the H1R and the distribution of H2R on
lymphocyte subsets in general, signaling through the H1R
was associated with enhancement and signaling through the
H2R with inhibition of lymphocyte responses. It has been
suggested by several studies that histamine and its deriva-
tives can inhibit the immune response by enhancing the ac-
tivity of T suppressor cells through H2R and natural sup-
pressor cells via H1R [321, 322]. The impacts of histamine
on T helper lymphocytes are differential and complex; see in
Fig. (11). T lymphocytes, mainly T helper lymphocytes, play
a significant role in the pathogenesis of atopic asthma.
Helper T lymphocytes can be divided into two subsets (T
helper type 1 cells (Th1) and Th2) based on their cytokine
profile and distinct functions and both the subsets play dis-
tinctive roles in the development, initiation, and regulation of
the immune response. Th1 cells were found to be responsive
in delayed type hypersensitivity (DTH) and cytotoxic re-
sponse, while Th2 cells were involved in allergic disease via
activating B-lymphocytes and regulating antibody (IgG and
IgE) secretion; see in Fig. (11). Th1 cells secrete important
cytokines as interleukin (IL)-2, IFN-, IL-3, and granulocyte
monocyte colony stimulating factor (GM-CSF), while Th2
cells secrete cytokines such as IL-3, IL-4, IL-5, IL-10, IL-13,
and GM-CSF. Histamine downregulates the proli