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INFLAMMATION AND IMMUNOPHARMACOLOGY
Johnson and Johnson Pharmaceutical Research and Development LLC, San Diego, California
Received for publication
November 5, 2002
Accepted
February 25, 2003.
 |
Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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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
-3-methoxyestra-1,3,5
(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122
[GenBank]
). 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.
). 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 (H4 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 H4 receptor in mouse, rat, and guinea pig were
cloned (Liu et al.,
2001b).
The amino acid sequence of the H4 receptor has low homology with
other histamine receptors. Its closest member in the histamine receptor family
is the H3 receptor that shares only a 35% amino acid homology with
the H4 receptor, although the homology in the transmembrane region
is 58%. However, the H4 receptor expression pattern is distinct
from the H3 receptor. Although the expression of the H3
receptor is mainly restricted to cells in the central nervous system
(Lovenberg et al., 2000), the
H4 receptor seems to be limited to cells of the hemopoietic lineage
(Oda et al., 2000;
Liu et al., 2001a;
Morse et al., 2001;
Zhu et al., 2001). The
expression of H4 receptors on hemopoietic cells is not unique among
the histamine receptor family, because T cells and dendritic cells also
express H1 and H2 receptors
(Jutel et al., 2001;
Szeberenyi et al., 2001).
Pharmacological properties of the H4 receptor have been revealed
using H4 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 H1 and H2 receptor antagonists and
agonists do not bind to the H4 receptor. However, more typical
H3 receptor ligands (such as thioperamide, clobenpropit, imetit,
and R--methylhistamine) could bind the H4 receptor
with affinities different from that of the H3 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 H1 receptors mediate this
action through Gq proteins, resulting in calcium mobilization,
H2 receptors signal through Gs proteins, and cAMP increase,
whereas H3 receptors signal through Gi/o proteins and
inhibition of cAMP (Lovenberg et al.,
1999). In the literature, two signaling pathways are thought to be
used by the H4 receptor. First, using cells transfected with the
H4 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
H3 receptor. Second, one study showed that histamine could not
alter cAMP levels in H4 receptor-transfected cells, but instead
increased calcium mobilization if the cells were cotransfected with
Gq/i1/2, Gq/i3, or G16 proteins
(Morse et al., 2001). The same
study showed that histamine induced mitogen-activated protein kinase
phosphorylation, which was inhibited by pertussis toxin (PTX). However,
signaling pathways mediated by endogenous H4 receptors have not
been studied.
Mast cells are important effector cells in allergic diseases. Mast cells bind IgE with IgE receptor, and subsequent contact with antigens will trigger IgE receptor cross-linking and the release of preformed mediators, such as serotonin and histamine, and de novo-produced mediators, such as prostaglandins and leukotrienes. Although mast cells are best known for their histamine-releasing capacity, little is known about the effect of histamine on mast cells themselves.
In the present study, the expression pattern of mouse H4 receptor on various purified hematopoietic cells and in various tissues was investigated. We showed that the mouse H4 receptor was expressed specifically on eosinophils and mast cells. Bone marrow-derived mast cells were used to study the functional aspects and signaling pathways of the endogenous mouse H4 receptor.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Thapsigargin, U73122 [GenBank] and U73343 [GenBank] were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Transwells were purchased from Costar (Cambridge, MA). LTB4 and prostaglandin detection kits were from Cayman Chemicals (Ann Arbor, MI). Fluo-3 was from TEF Labs (Austin, TX), and Pluronic acid was from Molecular Probes (Eugene, OR). Pertussis toxin and anti-DNP IgE was from ICN Pharmaceuticals (Costa Mesa, CA). All other antibodies were from BD PharMingen (San Diego, CA). Polylysine-coated black wall 96-well tissue culture plates were purchased from BD Biosciences (San Jose, CA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Methods
Generation of H4 Receptor Gene Knockout Mice (H4
R-/-). H4R-/- mice were generated by Lexicon Genetics
(Woodlands, TX). A 9-kb mouse genomic fragment containing the mouse
H4 receptor gene was obtained from the embryonic stem cell line 2G9
and was used as a template to prepare the knockout construct. A 0.5-kb region
covering 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. Homologous recombination in embryonic stem
cells was confirmed by Southern analysis using a 3' external probe
amplified from the mouse H4 receptor gene with oligonucleotides
5'-GAG ATG TAG ATG TGG TCG TTT G and 5'-CAT GTG CAG GCA CAC ACA
TAC. The Southern blot of ES cell DNA digested with NcoI produced a
4.9-kb wild-type band and a 3.4-kb targeted band
(Fig. 1). Chimeric mice were
generated from embryos injected with embryonic stem cells. Germline mice were
obtained by breeding chimeric male mice with C57BL/6 females. Germline mice
heterozygous for the disrupted H4 receptor gene were identified by
PCR. Wild-type and H4 R-/- mice were obtained from cross-breeding
of heterozygous mice.
|
Detection of Mouse H4 Receptor Expression. RNA from tissues and purified cells was prepared using a RNeasy kit according to the manufacturer's instructions. H4 receptor RNA was detected by RT-PCR using specific mouse H4 receptor primers (5'-ATG TCG GAG TCT AAC AGT ACT GG and 5'-AGA AGA TAC TGA CTG GTT CTG TGA). RT products of multiple tissues and cell types were amplified by PCR under conditions of 94°C 45 s, 55°C 45 s, and 72°C 2 min for 35 cycles. The PCR products were run on a 1% agarose gel with ethidium bromide (10 µg/ml), and DNA was visualized with UV light. The amplified mouse H4 receptor cDNA is 1185 bp in size.
Mouse Th1 cells, Th2 cells, Tc1 cells, Tc2 cells, B cells, and macrophages
were activated, and total RNA was prepared as described previously
(Shier et al., 2000).
Eosinophils were in vitro differentiated from C57BL/6J mouse bone marrows.
Bone marrow was aseptically isolated from the femurs. The cells (2 x
105/ml) were cultured at 37°C with 5% CO2 in RPMI
1640 culture medium consisting of 10% FCS, 0.1 mM nonessential amino acids, 50
µg/ml penicillin/streptomycin, 0.2 ng/ml IL-3, 0.4 ng/ml IL-5, and 0.2
ng/ml granulocyte-macrophage colony-stimulating factor. After 6 days, the
medium was refreshed and cells were cultured for seven more days. Cells were
stained with hematoxylin-eosin dyes and >95% of the cells displayed
eosinophil phenotype. Mouse kidney, liver, thymus, spleen, lung, and brain
were isolated from C57BL/6J mice. RNA was isolated from the tissues as
described above.
Total RNA samples (5 µg) were run on a RNA gel and then transferred
overnight to a nylon blot. The blot was prehybridized with ExpressHyb solution
for 30 min at 68°C. The mouse H4 receptor cDNA clone
(Liu et al., 2001a) and the
purified RT-PCR product of the mouse H3 receptor
(Liu et al., 2001b) were
labeled using the Rediprime II kit. The blot was hybridized for 2 h at
68°C, followed by one wash (2x standard saline citrate and 0.05%
SDS) of 40 min at room temperature, and a second wash (0.1x standard
saline citrate and 0.1% SDS) of 40 min at 50°C. The blots were exposed to
X-ray film at -70°C with two intensifying screens for 16 h.
Detection of Human H4 Receptor Expression. Human
basophils (98% purity) were isolated from human peripheral blood mononuclear
cells using a basophil enrichment kit. Human mast cells were differentiated
from human CD34+ cells purified from cord blood, using serum-free
medium supplemented with human stem cell factor (SCF) (100 ng/ml) and IL-6 (50
ng/ml) (Dahl et al., 2002).
Cells were grown for 12 to 14 weeks, and media supplemented with cytokines was
changed once a week. Cells were monitored weekly for their mast cell
properties, using Geimsa, toluidine blue, tryptase, and CD117 staining. The
cells showed metachromatic granule staining properties, as early as 2 to 3
weeks and by week 12, the purity of these cells for mast cell properties was
95%. Human mast cell line HMC-1 was cultured in Iscove's medium containing 10%
FCS, 2 mM glutamine, and 1.2 mM monothioglycerol. Total RNA was extracted from
human basophils, mast cells, and HMC-1 cells using an RNeasy kit, and 250 ng
of RNA was used for the RT reaction according to manufacturer's instructions.
PCR using human H4 receptor-specific primers (5'-ACT AGA ATT
CGC CAC CAT GCC AGA TAC TAA TAG CAC and 5'-ATG CAG GAT CCA GCA TTT GAG
ACT GAC AGG TAT) was carried out as described previously
(Liu et al., 2001a).
Bone Marrow Mast Cell Culture. Mast cells were differentiated from
bone marrows collected from H4 receptor gene knockout
(H4R-/-), H3 receptor gene knockout (H3R-/-)
mice (Toyota et al., 2002),
wild-type mice, and BALB/c and C57BL/6J mice. Bone marrow was aseptically
isolated from the femurs. The cells (5 x 105/ml) were
cultured at 37°C with 5% CO2 in RPMI 1640 culture medium
consisting 10% FCS, 0.1 mM nonessential amino acids, 50 µg/ml
penicillin/streptomycin, and 20% WEHI-3 conditioned medium. WEHI-3 cells were
cultured in Iscove's Dulbecco's medium with 10% FCS, 4 mM
L-glutamine, 1.5 g/l sodium carbonate, 0.05 µM
-mercaptoethanol, and 50 µg/ml penicillin/streptomycin. The filtrated
supernatant was used as WEHI-3 conditioned medium.
After 16-h culture, the nonadherent bone marrow cells were transferred to a new flask for further culture. The medium was refreshed once a week. After 4 weeks, the cells were analyzed by flow cytometry for IgE receptor and CD117 (c-kit), which are expressed specifically on mast cells. IgE receptor on mast cells were detected by incubating with anti-DNP IgE or vehicle for 30 min, followed by fluorescein isothiocyanate-labeled anti-IgE antibody. Mast cells were incubated with fluorescein isothiocyanate-labeled anti-CD117 antibody for 30 min on ice. The majority of the bone marrow cells was confirmed to be mast cells with >99% IgE receptor positive and >99% CD117 positive. Mast cells of 4 to 8 weeks were used for experiments. No difference in proliferation and in expression of IgE receptor or c-kit receptor was observed in mast cells derived from H3R-/-, H4R-/-, and wild-type mice.
Degranulation Assay. Mast cells (5 x 105/ml) were
sensitized overnight with 2 µg/ml anti-DNP IgE. Mast cells (2 x
105/well) were plated out in a 96-wells plate and incubated for 15
min with 10 µM histamine or vehicle at 37°C. Degranulation was achieved
by adding different concentrations of DNP-HSA for an additional 30 min. Total
release of mast cell contents was achieved by adding 1% Triton X-100. The
plates were spun down (1000 rpm, 5 min, 5°C), and supernatants were
analyzed for -hexosaminidase.
-Hexosaminidase was measured by adding 25 µl of supernatant to 50
µlof10mM p-nitrophenyl
N-acetyl--D-glucosaminide in 0.1 M sodium citrate
buffer (pH 4.5) for 2 h at 37°C. The reaction was stopped by adding 50
µl of 0.4 M glycine (pH 9). The plates were measured at wavelength 405 nm.
The percentage of degranulation was calculated as ((A -
B)/(T - B) x 100), where A is levels
of -hexosaminidase released from stimulated cells, B is that
released from unstimulated cells, and T is total content of the
cells.
To study the induction of leukotriene and prostaglandins, IgE-sensitized mast cells (1 x 106/well) were incubated for 15 min with 10 µM thioperamide or vehicle, followed by 30- or 210-min incubation with 10 µM histamine or 5 µg/ml compound 48/80. LTB4 and prostaglandin levels were measured in supernatants according to the manufacturer's instruction.
Chemotaxis Assay. Transwells with a pore size of 8 µm were coated with 100 µl of 100 ng/ml bovine fibronectin for 2 h at room temperature. After removal of the fibronectin, 600 µl of RPMI 1640 medium with 1% BSA in the presence of histamine (ranging from 1.25 to 20 µM) was added to the bottom chamber. Subsequently, 10 µM histamine receptor antagonists (diphenhydramine, ranitidine, thioperamide), U73122 [GenBank] (1.1, 3.3, and 10 µM), or U73433 [GenBank] (1.1, 3.3 and 10 µM) was added to the top and bottom chambers. Mast cells (2 x 105/well) were added to the top chamber. The plates were incubated for 3 h at 37°C. Transwells were removed, and the number of cells in the bottom chamber was counted for 1 min by flow cytometer. To study PTX effects, mast cells (1 x 106 cells/ml) were pretreated for 16 h with 0, 0.5, 5, or 50 ng/ml PTX. Cells were washed afterward and put in the upper chamber as described above.
Calcium Mobilization. Mast cells (2 x 105/well) were loaded with 4 µM calcium dye Fluo-3 (acetoxymethyl ester) in dye-loading buffer for 1 h at 37°C. The dye-loading buffer is RPMI 1640 medium without phenol red and contains 0.5% BSA, 2.5 mM probenecid, and 0.08% Pluronic acid. Cells were spun down and taken up in loading medium which is RPMI 1640 medium (without phenol red) containing 0.5% BSA. Cells were plated out in polylysine-coated black wall 96-well tissue culture plates. Before measurements, the plates were spun for 3 min at 1000 rpm at room temperature. Calcium mobilization was assayed in a fluorometric imaging plate reader 384 (Molecular Devices Corp., Sunnyvale, CA). The fluorescence intensity was calculated as the maximum minus the minimum fluorescence over a 2-min period. All data points were done in triplicates, and experiments were repeated at least three times with different batches of mast cells.
Histamine receptor agonists and antagonists were added to the cells 10 min before the calcium measurements. In calcium storage experiments, Fluo-3-loaded mast cells received a first addition of 3 mM EDTA or 10 µM thapsigargin or PBS. After stabilization of the signal, mast cells received a second addition of 10 µM histamine.
For PTX treatment, mast cells (1 x 106 cells/ml) were pretreated for 16 h with 0, 0.5, 5, or 50 ng/ml PTX. Cells were washed and loaded with Fluo-3 as described above. For phospholipase C (PLC) inhibitor treatment, mast cells were treated with 1.1, 3.3, and 10 µM U73122 [GenBank] or U73433 [GenBank] 10 min before stimulation with 10 µM histamine.
To detect calcium response triggered by IgE receptor cross-linking, mast cells (5 x 105/ml) were sensitized overnight with 2 µg/ml anti-DNP IgE. Cells were washed and loaded with Fluo-3 as described above. During the calcium measurements, 5 µM histamine was added followed 3 min later with different concentrations of DNP-HSA.
cAMP Measurements. Mast cells (1 x 106/well) in culture medium containing 1 µM 3-isobutyl-1-methylxanthine were plated out in a 96-well plate. Cells were incubated for 30 min at 37°C. Histamine receptor antagonist (10 µM) was added 15 min before histamine and/or 100 µM forskolin addition for 30 min at 37°C. Intracellular cAMP levels in cell lysates were determined using the Biotrack cAMP enzyme immunoassay system according to the manufacturer's instructions.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). In the
present study, mouse H4 expression was determined in different
tissues and purified cell types of the hemopoietic lineage. Abundant
expression of mouse H4 receptor was detected in untreated mast
cells and antigen-activated IgE-primed mast cells by RT-PCR
(Fig. 2A). In contrast,
H4 receptor was not detected in any of the tissues tested, such as
lymph nodes, kidney, liver, thymus, spleen, heart, lung, and brain. In
addition, expression was not detected in many different immune cell types,
including CD4+ effector Th1 and Th2 cells, CD8+ effector
Tc1 and Tc2 cells, resting and LPS-activated B cells, as well as
macrophages.
|
Expression of H4 receptor in mast cells and eosinophils was further confirmed by Northern blot analysis (Fig. 2, B and C). Both H1 and H2 receptors were detected on mast cells (data not shown), whereas H3 receptor was undetectable by Northern blot analysis (Fig. 2C, left) and RT-PCR (data not shown). Consistent with the expression profile in mice, H4 receptor was detected in human cord blood-derived mast cells and in human HMC-1 mast cell line by RT-PCR (Fig. 2D). In addition, human H4 receptor was expressed in basophils (Fig. 2D) but not in neutrophils (data not shown). In summary, our data show that the H4 receptor is expressed on mast cells, basophils, and eosinophils.
H4 Receptors Mediate Calcium Mobilization in Mast Cells. Histamine binding to its receptors activate G proteins, which result in changes of calcium or cAMP levels. Histamine induces a concentration-dependent increase of cAMP in mast cells (Fig. 3). This response was unaffected by H3/H4 antagonist thioperamide and H1 antagonist diphenhydramine (data not shown), thereby excluding a role for H1,H3,or H4 receptors. However, the H2 receptor antagonist ranitidine could inhibit the histamine-induced cAMP increase. The results indicate that the histamine-induced cAMP increase in mast cells is H2 receptor-mediated.
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Calcium mobilization was observed in mast cells induced by histamine in a
concentration-dependent manner (Fig.
4A). The response peaked at about 20 s after histamine addition
and returned to basal levels within 1 min
(Fig. 4A, inset). The
ED50 value of histamine-induced calcium mobilization was 3.8 µM.
Neither H1 receptor antagonists nor H2 receptor
antagonists altered the histamine-induced calcium mobilization
(Fig. 4B). However,
thioperamide (Fig. 4B)
inhibited the histamine-induced calcium mobilization in a
concentration-dependent manner, with an IC50 value of 1.00 ±
0.5 µM. This IC50 value is consistent with the relative binding
affinities of histamine and thioperamide
(Liu et al., 2001a). Together,
the data suggest that H3 and/or H4 receptors are
involved in calcium mobilization in mast cells.
|
Because mast cells do not express H3 receptors (Fig. 2C), it is likely that the calcium response is mediated by H4 receptors. A direct proof of H4 receptor-mediated calcium mobilization was demonstrated in mast cells generated from H4R-/- and H3R-/- mice. In contrast to wild-type mast cells, up to 30 µM histamine stimulation in H4R-/- mast cells did not result in calcium mobilization (Fig. 4C). Mast cells from H4R+/- mice showed an intermediate calcium response compared with mast cells from wild-type mice. This response is histamine-specific because H4R-/- mast cells mediated normal calcium responses to ATP or ionomycin (data not shown). Furthermore, H3R-/- mast cells showed a normal calcium response to histamine comparable with that in wild-type mast cells (data not shown). Therefore, it can be concluded that histamine induces calcium mobilization in mast cells via the H4 receptor.
H4 Receptors Trigger Calcium Release from Intracellular Calcium Stores. To determine the source of calcium in histamine-induced calcium mobilization, either EDTA or thapsigargin was used in experiments to deplete calcium from extracellular environment or intracellular calcium storage, respectively. Histamine-induced calcium mobilization was not affected by EDTA but was completely abolished by thapsigargin (Fig. 5, A and B). Thus, histamine mediates the release of calcium from intracellular calcium stores in mast cells.
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H4 Receptor Mediates Calcium Mobilization through Gi/o
Proteins and PLC. To determine the G proteins used by H4 receptor in mast
cells, Gi/o protein inhibitor PTX was used in experiments. Pretreatment
of mast cells with PTX inhibited the histamine-induced calcium response
completely (Fig. 5C), but the
calcium response toward ionomycin or ATP was unaffected (data not shown),
indicating that the PTX inhibitory effect is histamine-specific. Therefore, it
seems that Gi/o proteins are acting downstream of the H4
receptor, leading to calcium mobilization.
The possible involvement of PLC in histamine-induced calcium mobilization
was studied using the PLC inhibitor U73122
[GenBank]
and its inactive analog U73343
[GenBank]
(Thompson et al., 1991).
U73122
[GenBank]
inhibited the histamine-induced calcium mobilization in a
concentration-dependent manner with a complete inhibition at 10 µM, whereas
the inactive analog U73343
[GenBank]
(up to 10 µM) was unable to alter this response
(Fig. 5D). These results
indicate that the histamine effects on calcium mobilization involved PLC
activation.
Histamine Does Not Alter Degranulation through H4 Receptors. Effects of histamine on IgE receptor-mediated calcium response and degranulation in mast cells were investigated. IgE-primed mast cells were pretreated with histamine, followed by antigen stimulation. Histamine did not alter antigen-IgE triggered calcium mobilization (Fig. 6A). Antigen induced degranulation of IgE-primed mast cells from wild-type and H4R-/- mice was also unaffected by histamine pretreatment (Fig. 6B). In addition, thioperamide did not have any effects on antigen-IgE-mediated mast cell degranulation (data not shown).
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The effects of histamine on the production of de novo-synthesized mediators such as prostaglandins and leukotrienes were also investigated. Compound 48/80 induced LTB4 and prostaglandin release by mast cells, whereas histamine did not alter LTB4 or prostaglandin levels (Table 1). In summary, H4 receptor and histamine do not seem to be involved in antigen-induced degranulation because histamine does not induce degranulation nor is it involved in the de novo production of LTB4 and prostaglandins by mast cells.
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Histamine Mediates Chemotaxis through H4 Receptors. Chemotaxis of mast cells toward histamine was investigated using a Transwell system. Histamine induced mast cell migration in a concentration-dependent manner (Fig. 7A). This observed effect was due to chemotaxis but not chemokinesis, because cell migration was abolished when the histamine concentration gradient was disrupted. Thioperamide inhibited histamine-induced mast cells chemotaxis in a concentration-dependent manner, whereas neither diphenhydramine nor ranitidine had any effects (Fig. 7, B and C). The IC50 value of thioperamide in chemotaxis is similar to that in the calcium mobilization assay. To distinguish between H3 and H4 receptor-mediated effects on chemotaxis of mast cells, the chemotaxis assay was performed using mast cells derived from H4R-/- or H3R-/- mice. No migration of H4R-/- mast cells toward histamine was observed (Fig. 7A). In contrast, chemotaxis of H3R-/- mast cells to histamine was similar to that in wild-type mast cells (data not shown). Thus, histamine-induced chemotaxis of mast cells is mediated through the H4 receptor.
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H4 Receptor-Mediated Chemotaxis Involves Gi/o Proteins
and PLC. 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
(Fig. 7D). A complete
inhibition of histamine-induced chemotaxis was observed at 50 ng/ml, a
concentration with similar effects in inhibiting calcium mobilization. PLC was
also involved in the histamine-induced chemotaxis because U73122
[GenBank]
could inhibit
the chemotaxis in a concentration dependent fashion
(Fig. 7E), while its inactive
analog U73343
[GenBank]
did not alter the chemotaxis. Together, these results suggest
that the histamine-induced chemotaxis of mast cells involves Gi/o
proteins and PLC, similar to that of the calcium response.
|
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Liu et al., 2001a;
Morse et al., 2001).
Interestingly, this is the first study to show that mast cells express the
H4 receptor, but not the H3 receptor. In the literature,
it has been unclear whether mast cells express the H3 receptor.
Most studies used thioperamide as a specific H3 antagonist
(Kohno et al., 1994;
Bissonnette, 1996), but recent
data indicate that both the mouse and human H4 receptor can bind
thioperamide as well (Liu et al.,
2001b). It is therefore likely that the effects of histamine on
mast cells that can be blocked by thioperamide are actually mediated by
H4 receptors. The role of the H4 receptor in some of the physiological functions of mast cells was investigated. One of the major biological functions of mast cells is to release inflammatory mediators in response to antigens. The major mechanism of such release is through IgE-mediated degranulation. The present work shows that histamine does not seem to have any effects on degranulation, either on its own or in combination with antigen-IgE complexes. In addition, histamine does not seem to alter mast cell proliferation or survival (data not shown). Similarly, H4R-/- mast cells did not show any defects in degranulation, proliferation, or survival, indicating that the H4 receptor has no role in these processes.
Mast cell progenitor cells, which are present in the bone marrow, migrate
to connective or mucosal tissue where they differentiate into the mature form.
It is thought that chemoattractants such as stem cell factor might be
important for this localization. Migration of mast cells may also play a role
in allergic rhinitis and allergy where increases in mast cell number are found
(Kirby et al., 1987;
Crimi et al., 1991;
Amin et al., 2000;
Gauvreau et al., 2000;
Kassel et al., 2001). In
addition, it is known that in response to antigens there is a redistribution
of mast cells to the epithelial lining of the nasal mucosa
(Fokkens et al., 1992;
Slater et al., 1996). It is
possible that some of the redistribution that is seen in allergic conditions
may be mediated by histamine because it would be continually produced under
such circumstances. The data presented here show that histamine is a potent
chemoattractant for mast cells and that this chemotaxis is mediated via the
H4 receptor. Antagonists of the H4 receptor may
therefore be useful in the treatment of asthma or allergic rhinitis.
Currently, we are addressing these questions with in vivo models.
Using specific histamine receptor antagonists as well as mast cells derived
from H4R-/- and H3R-/- mice, we demonstrated that
histamine induced calcium mobilization from intracellular stores in mast cells
via the H4 receptor. Calcium mobilization via the H4
receptor has also been observed using cells cotransfected with both the
H4 receptor and chimeric G proteins
(Morse et al., 2001). However,
other studies using human H4 receptor-transfected cells have shown
that histamine activation of the cells resulted mainly in decreased cAMP
levels (Oda et al., 2000;
Liu et al., 2001a;
Zhu et al., 2001). Because
results of transfected cells depend on the endogenous machinery of the cell,
different cell types can yield different results. In the present study, these
complications are not present, because the endogenous H4 receptor
was studied. Previously, similar calcium mobilization induced by histamine was
demonstrated in human eosinophils, which have been shown to express the
H4 receptor (Raible et al.,
1994). The calcium mobilization was inhibited by thioperamide, the
dual H3/H4 receptor antagonist. Nevertheless,
R--methyl-histamine and N--methyl-histamine
were less potent than histamine in inducing calcium mobilization, which is
more consistent with their respective affinities for the H4
receptor than for the H3 receptor
(Raible et al., 1994).
Therefore, it is likely that this response is mediated by the H4
receptor and not the H3 receptor. Thus, the activation of
H4 receptor both on mast cells and eosinophils results in calcium
mobilization.
The signaling pathways activated by the H4 receptor have also
been studied. Both Gi/o proteins and PLC are involved because PTX,
which inactivates Gi/o proteins, and the PLC inhibitor U73122
[GenBank]
,
inhibited chemotaxis and calcium mobilization. Gi/o proteins do not
activate PLC, but G protein 
subunits can activate
PLC2/3 (Exton,
1996; Clapham and Neer,
1997; Rhee, 2001).
It is therefore possible that PLC2/3 is activated by the G
protein subunits that are dissociated from Gi/o proteins
when histamine binds to the H4 receptor. Other G protein-coupled
receptors have also been shown to signal via PLC and Gi/o
(Seebeck et al., 1998;
Zussman et al., 1998;
Yang et al., 2002). The
activation of PLC may lead to the release of inositol-1,4,5-triphosphate
(IP3). 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).
Compound 48/80 has been reported to elicit calcium response in mast cells
through Gi/o proteins, phosphatidylinositol 3-kinase, Src, and Syk
(Shefler and Sagi-Eisenberg,
2001). Stem cell factor is also known to induce calcium
mobilization in mast cells involving activation of Gi/o proteins,
phosphatidylinositol 3-kinase, p38 mitogen-activated protein, and
mitogen-activated protein kinase kinases
(Dastych et al., 1998;
Sundstrom et al., 2001).
We propose the following signaling pathway involved in histamine activation
of the H4 receptor (Fig.
8). Histamine binds to the H4 receptor on mast cells
and eosinophils and causes the activation of PTX-sensitive Gi/o
proteins. Possibly G protein subunits dissociated from
Gi/o proteins trigger the activation of PLC. PLC hydrolyzes
phosphatidylinositol 4,5-biphosphate to diacylglycerol and IP3.
IP3 activates a calcium channel in the endoplasmic reticulum,
possibly through an IP3 receptor to release calcium. The increased
calcium levels trigger currently unknown signaling pathways, which will cause
mast cell chemotaxis toward histamine.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PTX, pertussis toxin; H3R-/-,
H3 receptor gene knockout; H4R-/-, H4
receptor gene knockout; RT, reverse transcription; PCR, polymerase chain
reaction; bp, base pair(s); Th, T helper; Tc, T-cytotoxic cells; FCS, fetal
calf serum; IL, interleukin; LTB4, leukotriene B4; BSA,
bovine serum albumin; PBS, phosphate-buffered saline; DNP-HSA, dinitrophenyl
human serum albumin; IP3, inositol 1,4,5-triphosphate; U-73122,
1-[6-[[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione;
U-73343,
1-[6-[[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione.
1 Current address: NV Organon, Molenstraat 110, P.O. Box 20, 5340 BH Oss, The
Netherlands. E-mail:
claudia.hofstra{at}organon.com 
Address correspondence to: Dr. Wai-Ping Fung-Leung, Johnson and Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121. E-mail: wleung{at}prdus.jnj.com
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