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DOI: 10.1002/eji.201343838

Eur. J. Immunol. 2014. 44: 204–214

Histamine synthesis is required for granule maturationin murine mast cells
Shunsuke Nakazawa∗1 , Mariko Sakanaka∗2 , Kazuyuki Furuta3 , Mayuko Natsuhara2 ,Hirotsugu Takano1 , Soken Tsuchiya4 , Yasushi Okuno5 , Hiroshi Ohtsu6 , Masahiro Nishibori7 ,Robin L. Thurmond8 , Noriyasu Hirasawa9 , Kazuhisa Nakayama1 , Atsushi Ichikawa10 ,Yukihiko Sugimoto4 and Satoshi Tanaka3
1

Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University,
Nishinomiya, Hyogo, Japan3
Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,
Okayama, Japan
4
Department of Pharmaceutical Biochemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto,Japan
5
Department of Systems Bioscience for Drug Discovery, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto,
Japan
6
Laboratory of Applied Quantum Medical Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan
7
Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,
Okayama, Japan
8
Janssen Research & Development, LLC., San Diego, CA, USA
9Laboratory of Pharmacotherapy, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan10
Institute for Biosciences, Mukogawa Women’s University, Nishinomiya, Hyogo, Japan
2

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase(HDC). Previous studies have shown that Hdc−/− mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether thehistamine synthesized in mast cells regulates the granule maturation of murine mast
cells. Several genes, including those encoding granule proteases and enzymes involved
in heparin biosynthesis, were downregulated in Hdc−/− peritoneal mast cells. Impaired
granule maturation was also found in Hdc−/− BM-derived cultured mast cells when they
were cocultured with fibroblasts in the presence of c-kit ligand. Exogenous applicationof histamine and several H4 receptor agonists restored the granule maturation of Hdc−/−
cultured mast cells. However, the maturation of granules was largely normal in Hrh4−/−
peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitorof vesicular monoamine transporter-2, did not affect granule maturation. In vivo experiments with mast cell deficient KitW /KitW-v mice indicated that the expression of the
Hdc gene in mast cells is required for granule maturation. These results suggest that
histamine promotes granule maturation in mast cells and acts as an proinflammatory
mediator.

Keywords: Cell differentiation r Histamine r Inflammation r Mast cell
See accompanying Commentary by Hallgren and Gurish

Correspondence: Prof. Satoshi Tanaka
e-mail: tanaka@pharm.okayama-u.ac.jp

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim∗

These authors contributed equally to this study.

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Innate immunity

Introduction
Histamine plays a critical role in various physiological responses,
such as inflammation, gastric acid secretion, neurotransmission,and immune modulation, which are mediated by its specific receptors, the H1 , H2 , H3 , and H4 subtypes [1–4]. Mast cells are the primary sources of tissue histamine and it is liberated from the cytoplasmic granules upon various stimuli including IgE-dependent
Ag stimulation [5]. Histamine synthesis is mediated by L-histidine
decarboxylase (HDC), which is initially synthesized as a 74 kDa
precursor and then is in posttranslationally modified [6]. The
74 kDa HDC is mainly localized in the cytosol, while the processed
53 kDa form is distributed to the granules in mast cells and activated neutrophils [6, 7]. In a mouse mastocytoma P-815 cells,
caspase-9-mediated cleavage results in enzymatic activation of
HDC [8].
We have explored novel functions of histamine using a mousestrain lacking the Hdc gene. In the first report of Hdc−/− mice,
we noticed that peritoneal mast cells (pMCs) and skin mast cells
exhibited an aberrant morphology with severely decreased granule contents [9]. Although several studies indicated that mast cells
express specific histamine receptors, it remains unknown how histamine affects the nature of mast cells. On the other hand, positive
modulators for growth and differentiation of mast cells, such as
IL-3 and c-kit ligand (Kitl), were found to induce a transient
increase in histamine synthesis [10, 11], raising a possibility that
histamine is involved in the process of mast cell maturation.
We hypothesized that newly synthesized histamine may play a
critical role in granule maturation of mast cells in an autocrine
fashion. We, here, took two different approaches to investigate
the mechanism underlying the histamine-mediated promotion of
granule maturation of mast cells; an in vitro approach using amast cell fibroblast coculture model and an in vivo one using themast cell reconstitution system with mast cell deficient KitW /KitW-v
mice. This study proposes a novel function of histamine in mastcell biology.

Results−/−

Characterization of the pMCs in Hdc

mice

We previously reported that the cutaneous and pMCs of Hdc−/−
mice demonstrate aberrant granules with severely decreased
contents [9]. pMCs in Hdc−/− mice were poorly-stained with
Safranin-O and acidic Toluidine blue, compared with those in WT
mice (Fig. 1A). We found no significant differences in the number of total peritoneal cells and in the percentage of mast cells
based on the May–Grünwald–Giemsa staining between WT and
Hdc−/− mice (Fig. 1B and data not shown). Histamine was notdetectable in Hdc−/− peritoneal cells (Fig. 1C). Enzymatic activities of granule proteases, such as chymase, tryptase, and mast cell
carboxypeptidase A (MC-CPA), were found drastically decreased
in Hdc−/− peritoneal cells (Fig. 1D–F); the decrease is compatible with the electron-lucent appearance of granules ofC 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Characterization of Hdc−/− peritoneal mast cells (pMCs).
(A) Peritoneal cells collected from WT (+/+) and Hdc−/− (−/−) mice were
stained with Safranin-O (upper panels) or with acidic Toluidine blue
(lower panels); bar = 10 μm. (B) Percentages of mast cells of WT (white)and Hdc−/− (black) mice are determined based on the May–Grünwald–
Giemsa staining. (C) Histamine contents of peritoneal cells of WT(open column) and Hdc−/− mice were presented. N.D., not detectable.
(D–F) Enzymatic activities of (D) chymase, (E) tryptase, and (F) MCCPA in pMCs of WT (open columns) and Hdc−/− (closed columns) mice
were measured. (G) Gene expression profiles in BMMCs or in pMCs
were compared between WT and Hdc−/− mice. Downregulated genes
in the Hdc−/− mast cells (BMMCs or pMCs), were represented by green
columns, whereas upregulated genes were by red columns. The numberof the genes classified into each category is indicated. This is a representative of three independent experiments performed. (H) Expressionof granule protease genes, such as Mcpt4, Mcpt5, Mcpt6, Mcpt7, and Ctsg,
in the purified pMCs was compared through quantitative RT-PCR analyses based on the expression of Gapdh. (A–H) The values are represented
as the means + SEM and are representative of (A–F) 5 and (H) 3 independent experiments. The values of *p < 0.05 and **p < 0.01 are regarded
as significant by Student’s t-test.

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Figure 2. Characterization of Hdc−/−
BMMCs. (A) Surface expression levels ofc-kit and FcεRI in WT (+/+) and Hdc−/− (−/−)
BMMCs were measured by flow cytometry.
This is a representative of three experiments with similar results. (B) BMMCs
prepared from WT (white circles) or Hdc−/−(black circles) mice were sensitized with
anti-TNP IgE (clone IgE-3, 1 μg/mL) for 24
h and then stimulated with the indicatedconcentrations of TNP-BSA for 30 min.
Degranulation of mast cells were determined by measuring β-hexosaminidase
activity. Thapsigargin (Thg, 300 nM) wasused as a positive control. (C–E) BMMCs
sensitized with IgE as described above
were stimulated with the indicated concentrations of TNP-BSA for 3 h. The levelsof various cytokine (C, IL-6; D, TNF-α; andE, MCP-1) released into the medium were
measured. (F and G) Expression levels of H2(F) and H4 (G) receptors in BMMCs preparedfrom WT (+/+) and Hdc−/− (−/−) mice andpMCs from WT mice (pMC) were determined by quantitative RT-PCR analyses.
The expression levels were normalizedbased on those of GAPDH and presented as
the relative expression levels (H2 receptor,
the expression level in mouse stomach =1.0, and H4 receptor, the expression levelin mouse spleen = 1.0). (A–G) The values
are represented as the means + SEM andare representative of three independent
experiments. N.D., not detectable.

Hdc−/− mast cells, since neutral proteases account for a large partof constituents of mast cell granules.
We then compared gene expression profiles in IL-3-dependent
BM-derived cultured mast cells (BMMCs), and those in mature
mast cells purified from the peritoneal cavity between WT and
Hdc−/− mice (Fig. 1G); the former cells have been used as one of
the common culture models of mast cells. The expression levelsof 170 genes were changed greater than twofold between WTand Hdc−/− mast cells. Among them, the expression levels of 158
genes were changed between WT and Hdc−/− pMCs, whereas theexpression levels of only 15 genes were changed between WTand Hdc−/− BMMCs. The genes encoding granule proteases, suchas Mcpt4, 5, 6, 7, and cathepsin G (Ctsg), and enzymes involved
in heparin biosynthesis, such as Ext1, Ndst2, Hs2st1, and Hs3st1,
were downregulated in Hdc−/− pMCs, in good agreement with
impaired granule maturation observed in Hdc−/− pMCs. Because
we could not detect any significant changes in mRNA expression ofseveral granule proteases with semiquantitative RT-PCR analysis
previously [9], quantitative RT-PCR analysis was performed in thisstudy. Expression of granule protease genes, such as Mcpt4, 5, 6, 7,
and Ctsg, was found to be decreased (<30% of the WT) in Hdc−/−
pMCs (Fig. 1H). These findings raised a possibility that histamine
affects the function of master transcription factors that regulates
the terminal differentiation of mast cells. Impaired expression ofhematopoietic transcription factors, such as Gfi1, Gata2, and Myb,C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

in Hdc−/− pMCs, makes it possible that they are involved in theprocess of granule maturation.

Characterization of Hdc−/− BMMCs
As described above, comparison of gene expression profiles(Fig. 1G) revealed that the absence of HDC has a lesser impact
on BMMCs than on pMCs. Surface expression levels of c-kit andFcεRI were similar in WT and Hdc−/− BMMCs (Fig. 2A). The
levels of degranulation and production of inflammatory cytokines,
such as IL-6, TNF-α, and MCP-1, of Hdc−/− BMMCs upon IgEmediated Ag stimulation were also comparable to those of WT
BMMCs (Fig. 2B–E). Quantitative RT-PCR analyses revealed that
BMMCs expressed both histamine H2 and H4 receptor subtypes attranscriptional levels whereas peritoneal mast cells did only the H4
subtype (Fig. 2F and G). No significant changes were detected inmRNA levels of the H2 and H4 subtypes between WT and Hdc−/−
BMMCs. H1 and H3 receptor mRNAs were under the detectionlevels in both BMMCs and peritoneal mast cells (data not shown).

Impaired granule maturation of Hdc−/− CTMC like
cultured mast cells
We have recently established a culture model that can reproduce
at least in part the process of maturation of connective tissue-type
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Figure 3. Restoration of granule maturation in Hdc−/− cultured mast cells by exogenous histamine. (A) BMMCs prepared from WT (+/+) andHdc−/− (−/−) mice were cocultured with mitomycin C-treated Swiss 3T3 fibroblasts in the presence of 30 ng/mL Kitl. The cells were visualized
by Alcian blue/Safranin-O staining at indicated time points; bar = 10 μm. (B) WT BMMCs (white columns) were cocultured as described above in
the absence (none) or presence of 3 μM tetrabenazine (Tbz) for 16 days, while Hdc−/− BMMCs (black columns) were cocultured in the absence orpresence of the indicated concentrations of histamine (histamine, 0, 10, 100 μM), 3 μM dimaprit (Dim), 3 μM clobenpropit (Clo), or 10 μM VUF8430(VUF). The number of Safranin-positive cells were counted at day 0 (D0) and day 16 (D16). (C) WT BMMCs were cocultured as described above in
the absence (white circles) or presence (white squares) of 3 μM tetrabenazine. Hdc−/− BMMCs were cocultured in the absence (black circles) orpresence of histamine (10 μM, black triangles, and 100 μM, black squares). (D and E) Expression levels of H2 (D) and H4 (E) receptor mRNAs in
cocultured BMMCs prepared from WT and Hdc−/− mice were determined by quantitative RT-PCR analyses. The expression levels were normalizedbased on those of GAPDH and represented as the relative expression levels (H2 receptor, the expression level in mouse stomach = 1.0, and H4
receptor, the expression level in mouse spleen = 1.0). (F–H) WT and Hdc−/− BMMCs were cocultured as described above in the absence or presenceof histamine (10 μM, and 100 μM). Granule protease activities (F, chymotryptic activity; G, tryptic activity; and H, CPA activity) at day 0 (D0) andday 16 (D16) were measured. (I) The cocultured cells (day 16) prepared from WT (white circles) or Hdc−/− (black circles) BMMCs were sensitizedwith anti-TNP IgE (clone IgE-3, 1 μg/mL) for 6 h and then stimulated with the indicated concentrations of TNP-BSA for 30 min. Degranulation ofmast cells was determined by measuring β-hexosaminidase activity. Thapsigargin (Thg, 300 nM) and compound 48/80 (C48/80, 10 μg/mL) wereused as positive controls. The values are presented as the means ± SEM and are representative of (B, C, F–H) 5 and (D, E, I) three independent
experiments. The values of *p < 0.05 and **p < 0.01 are regarded as significant by one-way ANOVA followed by Dunnett multiple comparison test
(D–H) or Tukey–Kramer multiple comparison test (B).

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mast cells (CTMCs); in this model, BMMCs are cocultured withmitomycin C-treated Swiss 3T3 fibroblasts in the presence ofKitl [12]. Induction of HDC during the coculture period raised apossibility that transiently induced histamine synthesis results in
granule maturation of mast cells. The enzymatic activity of HDC
was transiently but significantly increased in the early phase (day0, 3.37 ± 0.646, and day 4, 20.7 ± 1.68 pmol/min/mg protein,n = 5). The number of Safranin-positive cells was increased
during the coculture period in WT cells. However, such anincrease was significantly impaired in Hdc−/− cells (Fig. 3A and
B). Exogenously added histamine significantly augmented thenumber of Safranin-positive Hdc−/− cultured mast cells (Fig. 3B).
Because histamine synthesis was undetectable in Swiss 3T3
fibroblasts and FBS used in this study contained only a trace
amount of histamine (<50 nM), these results indicate that mastcell derived histamine is responsible for the increase in thenumber of Safranin-positive cells.
We then measured histamine contents in the cocultured mast
cells. Although the amount of cellular histamine in Hdc−/− mast
cells was under the detection level when cultured in medium
containing 10 μM histamine, the relatively higher levels ofhistamine, which were comparable to those in WT mast cells,
were detected in the Hdc−/− mast cells cultured in the presenceof 100 μM histamine (Fig. 3C). These results are reminiscent of
the previous studies that showed influx of extracellular histamine
through organic cation transporter(s) (OCT) with low affinity andhigh capacity for histamine, such as OCT-3 [13,14]. An increase in
the cytosolic histamine might result in accumulation of histaminein the granules. It is conceivable that the lack of histamine may
affect the electrostatic interactions among the contents of the
granules, such as those between sulfated proteoglycans andgranule proteases. Previous studies demonstrated colocalizationof heparin and histamine in mast cell granules and characterized
the electrostatic interaction between them [15]. However,tetrabenazine, which depleted histamine storage in cultured mast
cells, failed to suppress the increase in the number of Safraninpositive cells during the coculture period, indicating that storageof histamine in granules is not essential for their maturation(Fig. 3B and C).
We then explored another possibility that histamine enhances
granule maturation through acting on the surface receptors.
The increase in the number of Safranin-positive cells induced byhistamine was mimicked by clobenpropit, which is known to act asboth an H3 antagonist and an H4 agonist, and by VUF8430, which
is an H3 /H4 dual agonist, but not in the presence of dimaprit,an H2 /H4 dual agonist (Fig. 3B). Since BMMCs were found to
express H2 and H4 receptors (Fig. 2F and G), we investigated thetime course of mRNA expression of these receptors during thecoculture period and found that expression levels of these receptors in Hdc−/− cultured mast cells were not significantly different
from those in the WT cells (Fig. 3D and E). The different effects
among the various H4 agonists on the granule staining suggests
the involvement of the H2 receptor in histamine-mediated granulematuration in Hdc−/− mast cells [16–18]. However, no significant
increase in cAMP levels were detected in BMMCs stimulated with
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histamine or dimaprit (data not shown), indicating that the H2
receptor subtype plays no relevant roles.
Induction of chymotryptic activity during the coculture period
was also impaired in Hdc−/− mast cells, while exogenously added
histamine significantly restored the induction (Fig. 3F). In contrast
to the impaired expression of tryptase in Hdc−/− pMCs, inductionof tryptic activity during the coculture period was not affected by
the absence of HDC (Fig. 3G). Impaired induction in Hdc−/− mast
cells was also observed in MC-CPA activity but the induction of
the enzymatic activity was not restored in the presence of exogenous histamine (Fig. 3H). These results suggest that this culture
model should not reflect the endogenous action of histamine on
the expression of tryptase and MC-CPA.
Although Safranin-positive population and granule proteaseactivities, such as chymase and MC-CPA, were decreased in Hdc−/−
cultured mast cells, degranulation induced by IgE-mediated Ag
stimulation or by secretagogues such as thapsigargin and compound 48/80, remained unchanged (Fig. 3I), indicating that theabsence of HDC should not affect the FcεRI signaling pathway ordegranulation machinery in mast cells. Ag-induced production ofproinflammatory cytokines, such as IL-6, TNF-α, and MCP-1, wasseverely downregulated to the undetectable level by the coculture
in both the WT and Hdc−/− mast cells (data not shown).

Characterization of the histamine H4 receptor
expressed in mast cells
We then characterized the histamine receptors expressed in
BMMCs. Because the H4 subtype evokes Ca2+ mobilization by coupling with a trimeric G protein, Gi [19], we first measured changes
in the cytosolic Ca2+ concentrations in response to histamine.
When treated with histamine, both WT and Hdc−/− BMMCs clearly
exhibited a dose-dependent increase in cytosolic Ca2+ concentrations (Fig. 4A). The histamine-induced Ca2+ mobilization was
abolished by pretreatment of BMMCs with a specific H4 antagonist, JNJ7777120, but not with an H1 antagonist, pyrilamine, orwith an H2 antagonist, cimetidine (Fig. 4B), indicating that the H4
subtype is responsible for the histamine-induced Ca2+ mobilization in BMMCs. Histamine receptor ligands with an H4 agonistic
activity, such as clobenpropit, VUF8430, and dimaprit, all evoked
detectable Ca2+ mobilization in Hdc−/− BMMCs (Fig. 4C).
We then compared pMCs from Hrh4−/− mice with those from
WT mice. However, we unexpectedly failed to find any significant
differences in granule staining and in the number of pMCs
between WT and Hrh4−/− mice (Fig. 5A and B). On the other
hand, the histamine content in peritoneal cells of Hrh4−/− mice
was slightly but significantly lower than that of WT mice (Fig. 5C).
In Hrh4−/− pMCs, MC-CPA activity was significantly decreased
compared with that in WT mice, although an enzymatic activityof chymase or tryptase was not changed (Fig. 5D–F). An increasein the number of Safranin-positive cells observed during thecoculture period in the presence of the H4 agonists (Fig. 3B) raised
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Figure 5. Characterization of Hrh4−/− pMCs. (A) Peritoneal cells collected from WT (+/+) and Hrh4−/− (−/−) mice were stained with
Safranin-O (upper panels) or with acidic Toluidine blue (lower panels); bar = 10 μm. (B) Percentages of mast cells of WT (white column)
and Hdc−/− (black column) mice are determined based on the May–
Grünwald–Giemsa staining. (C) Histamine contents of peritoneal cellsof WT and Hrh4−/− mice were measured. (D–F) Enzymatic activities ofchymase, tryptase, and CPA in pMCs of WT (open columns) and Hrh4−/−(closed columns) mice were measured. (B–F) The values are represented
as the means + SEM and are representative of five independent experiments. The value of *p < 0.05 is regarded as significant by Student’s
t-test.

Figure 4. Ca2+ mobilization induced by histamine in BMMCs. (A) WT(+/+) and Hdc−/− (−/−) BMMCs were stimulated with the indicated concentrations of histamine (micromolar). Ca2+ mobilization induced bythrombin (Thr, 10 U/mL) was measured as a positive control. (B) WTand Hdc−/− BMMCs pretreated without (none) or with pyrilamine (Pyr,3 μM), cimetidine (Cim, 3 μM), or JNJ7777120 (JNJ, 3 μM) for 45 min
were stimulated with 3 μM histamine at the time point indicated byan arrow. (C) Hdc−/− BMMCs were stimulated with clobenpropit (Clo,30 μM), VUF8430 (VUF, 30 μM), or dimaprit (Dim, 30 μM) at the time
point indicated by an arrow.

of the proteoglycan sulfation. However, unlike Hdc−/− pMCs
(Fig. 1A), the granule staining was not impaired in Hrh4−/− mice
(Fig. 5A), suggesting that the H4 subtype may not be the primarytarget of histamine with respect to granule maturation. However,
MC-CPA activity was decreased in both the Hdc−/− and Hrh4−/−
pMCs as described above (Figs. 1F and 5F), indicating that the H4
subtype is responsible, at least in part, for MC-CPA expression.

Impaired granule maturation of Hdc−/− BMMCs
reconstituted in peritoneal cavity of KitW /KitW-v mice
Exogenous histamine-mediated restoration of Safranin-O staining in the coculture system strongly indicated that histamine candirectly promote granule maturation of mast cells in an autocrineC 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

fashion. However, there remained a possibility that histamine produced by nonmast cells could promote the granule maturationin vivo. We therefore investigated the role of HDC expressed inmast cells in granule maturation using a reconstitution system, inwhich BMMCs were engrafted into the peritoneal cavity of mastcell deficient, KitW /KitW-v mice. Five weeks after the engraftment,
the Hdc−/− mast cells were marginally stained, whereas the WT
mast cells exhibited a similar staining pattern to that of pMCs
in the mast cell sufficient control mice (Fig. 6A). The contrast
in the staining property was much more pronounced 10 weeks
after the engraftment. Differential counting of mast cells basedon the Alcian blue/Safranin-O staining revealed an increase in
the number of mast cells with abnormal staining properties in theperitoneal cavity engrafted with Hdc−/− BMMCs (Fig. 6B). The
percentages of mast cells in the total peritoneal cells were notsignificantly different between the mice engrafted with WT and
Hdc−/− BMMCs, in which no differences in the number of total
cells were observed in the peritoneal cavity (Fig. 6C and data
not shown), indicating that HDC is not implicated in colonization of the engrafted mast cells. The engraftment of WT BMMCs
restored the histamine content in the peritoneal cells, whereasthat of Hdc−/− BMMCs did not (Fig. 6D). Enzymatic activities of
granule proteases, such as chymase, tryptase, and MC-CPA, were
all significantly lower in the peritoneal cells of the mice engraftedwith Hdc−/− BMMCs than in those with WT BMMCs (Fig. 6E–G).
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Figure 6. Impaired granule maturation in Hdc−/− mast cells reconstituted in the peritoneal cavity of mast cell deficient, KitW /KitW-v mice. BMMCs
prepared from WT (+/+) or Hdc−/− (−/−) mice were engrafted into the peritoneal cavity of mast cell deficient, KitW /KitW-v mice (W/WV ). Peritoneal
cells were collected from the KitW /KitW-v mice 5 or 10 weeks after the engraftment or from the mast cell sufficient, Kit+ /Kit+ mice (WT). (A) The
peritoneal cells were collected and visualized by Safranin-O (top), acidic Toluidine blue (middle), or May–Grünwald–Giemsa (bottom) staining;bar = 10 μm. (B) The peritoneal cells were classified into four categories according to the staining property. Typical staining patterns are shown;bar = 10 μm. (C) Percentages of mast cells are determined based on the May–Grünwald–Giemsa staining. (D) Histamine contents in the peritoneal
cells were measured. N.D., not detectable. (E–G) Enzymatic activities of chymase (E), tryptase (F), and MC-CPA (G) in the peritoneal cells were
measured. (A–G) The values are represented as the means + SEM (WT, n = 5, W/WV —none, n = 3, W/WV —reconstituted, n = 7, each). The valuesof *p < 0.05 and **p < 0.01 are regarded as significant (versus W/WV —+/+) by Student’s t-test.

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pMCs in Hdc−/− mice (Fig. 1) and suggest that HDC expression inmast cells plays a critical role in their granule maturation.

Discussion
In this study, we demonstrated that histamine synthesis in mast
cells is required for their granule maturation. Although the previous study indicated the involvement of histamine in granule maturation of mast cells, it remained obscure how histamine should
promote the maturation. Accumulating lines of evidence have suggested that newly synthesized histamine in the cytosol follows
two fates; one is mediated by vesicular histamine transporters,
including vesicular monoamine transporter-2, which contributeto granule storage of histamine, and the other is by surface OCTs,
including OCT-3, which excrete excess amount of cytosolic histamine [13, 14, 20]. Granule maturation of mast cells, therefore,
could be enhanced by histamine found in three different sources;
namely, (i) granule histamine, (ii) extracellular histamine, and(iii) cytosolic histamine. It is not likely that granule histamine
affects the granule maturation of mast cells, because depletion of
granule histamine by treatment with tetrabenazine had no obvious effects on the granule staining. The functional H4 receptor
was found to express in murine mast cells and several kinds of theH4 agonists mimicked the effects of histamine, indicating the positive roles of the H4 receptor in the granule maturation. However,
granule maturation of the Hrh4−/− pMCs was found to be largely
intact, excluding the possibility that the H4 subtype play criticalroles in granule maturation. These findings highlight a possibility
that cytosolic histamine is involved in granule maturation of mast
cells. Schneider et al. reported that cytosolic histamine, which isincorporated into cells through OCT-3 when excess amount of
histamine is present in the extracellular spaces, inhibits the induction of HDC and several cytokines in murine IL-3-dependent BMderived basophils [13], although the exact intracellular targetsof histamine remains unknown. Very recently, covalent modification of heterotrimeric and low molecular weight G proteins by
histamine (histaminylation) has been detected in a mouse mastocytoma cell line, P-815 [21]. This modification is catalyzed bytransglutaminase-2 (TGM2). Since histamine synthesis was transiently induced and Tgm2 is upregulated during the coculture
period in our system [12], it is possible that histaminylation of thecytosolic proteins by TGM-2, which can target and activate Gαo1
and Cdc42, is involved in granule maturation of mast cells.
Aberrant granule formation has also been found in mast cells
derived from mice lacking Ndst2 or Srgn [22–24]; the former
encodes an enzyme involved in heparan sulfate biosynthesis andthe latter encodes a core protein of hematopoietic cell granuleproteoglycans. However, mature mast cells of these gene targeting mice harbored a small number of large low-density granules,
in contrast to the normal size and number of granules in Hdc−/−
mature mast cells. Ndst2−/− and Srgn−/− mast cells shared several
characteristics, such as decreased storage of granule proteases
with Hdc−/− mast cells and it was concluded that major defects
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at the posttranslational levels; impaired accumulation of sulfatedproteoglycans might lead to instability of the granule proteases in
Ndst2−/− and Srgn−/− mast cells. Histamine contents in pMCsof Hdst2−/− and Srgn−/− mice were found to be significantly
decreased [23,25], although it remains unknown whether expression of the Hdc gene is suppressed or granule storage of histamine
is impaired in these cells due to the absence of sulfated proteoglycans. A decrease in histamine synthesis may also be involvedin the aberrant granule maturation in Hdst2−/− and Srgn−/− mast
cells. Very recently, serious failure in granule maturation of mast
cells has also been found in Pla2G3−/− mice, in which significant
decreases in histamine synthesis and storage were observed inmast cells [26]. The impaired granule maturation of Pla2G3−/−
mast cells were also reproduced in the similar coculture system.
Since Hdc−/− mast cells are deficient in expression of multiple
granule proteases in addition to the absence of histamine, weshould give a cautious interpretation to the phenotypes observed
in Hdc−/− mice, as well as those in Ndst2−/− and Srgn−/− mice.
Since accumulating evidence suggests that a series of granule proteases are also involved in various immune responses [27], theroles of histamine may be overestimated in the previous studies
using Hdc−/− mice.
Several murine IgE clones have a potential to induce cytokine
production in the absence of the Ag and promote survival andmaturation of immature mast cells [28]. We previously reported
that histamine synthesis is transiently induced in BMMCs by IgE
in an Ag-independent manner [29]. Increased IgE levels, which
are often observed during chronic allergic diseases, were foundto upregulate the surface expression of FcεRI and to lead to exacerbation of the allergic diseases [30]. IgE-mediated exacerbation
may be mediated, at least in part, by self-reinforced mast cells.
Elevated histamine levels were also observed in various humanchronic inflammatory diseases, such as atopic dermatitis, chronic
urticaria, allergic asthma, multiple sclerosis, rheumatoid arthritis,and psoriasis [1]. Histamine may have a far greater influence on
the vicious cycle observed in chronic inflammatory responses than
expected. A strategy of inhibition of local histamine synthesis may
have clinical potentials for chronic inflammatory diseases, which
are characterized by accumulation of immature mast cells.

Materials and methods
AnimalsHdc−/− mice, which were originally generated with a mixed
genetic background of 129/Sv and CD1 [9], were backcrossed
to BALB/c strain or C57BL6 strain for 8–10 generations. Hrh4−/−
mice were generated by Lexicon Genetics, Inc. (The Woods, TX)
and were backcrossed to C57BL6 strain for 8–10 generations[19]. Specific pathogen-free, 8–12 week-old male and female
Balb/c mice and 5-week-old male WBB6F1-KitW /KitW-v mice wereobtained from Japan SLC (Hamamatsu, Japan), and all mice were
kept in a specific pathogen-free animal facility at Kyoto University
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Shunsuke Nakazawa et al.

and Okayama University. This study was approved by the Committee on Animal Experiments of Kyoto University, Mukogawa
Women’s University, and Okayama University.

Eur. J. Immunol. 2014. 44: 204–214

Measurement of cytokine release
Released cytokines (IL-6, TNF-α, and MCP-1) were measured by
ELISA kits (OptEIA, BD Biosciences, San Diego, CA) according to
the manufacturer’s instructions, respectively.

Materials
The following materials were commercially obtained from thesources indicated: HistodenzTM , p-nitrophenyl-β-D-2-acetoamide2-deoxyglucopyranoside, compound 48/80, and JNJ 7777120
from Sigma-Aldrich (St. Louis, MO), tetrabenazine, cimetidine,clobenpropit, dimaprit, pyrilamine, and VUF8430 from TOCRIS
Bioscience (Bristol, UK), recombinant mouse IL-3 from R&D Systems (Minneapolis, MN), S-2586 and S-2288 from Cromogenix(Milano, Italy), M-2245 from Bachem (Bubendorf, Switzerland),and Fura-2/AM from Dojindo Laboratories (Kumamoto, Japan).
All other chemicals were commercial products of reagent grade.

Preparation of BMMCs and CTMC-like MCs
BMMCs were prepared as previously described [12]. Briefly,
BM cells were cultured in the presence of 10 ng/mL IL-3 for 30–35
days to obtain mature BMMCs. Maturation of BMMCs were evaluated by the surface expression levels of FcεRI and c-kit and by
granule maturation based on the acidic Toluidine blue staining. A
CTMC culture model was originally established through cocultureof BMMCs with Swiss 3T3 fibroblasts [33, 34]. We modified thisculture method and obtained CTMC-like cultured mast cells using
mitomycin C-treated Swiss 3T3 fibroblasts in the presence of Kitl
(30 ng/mL) as previously described [12].

Purification of pMCs
pMCs (>95% purity, confirmed by Alcian blue/Safranin-O and
May–Grünwald–Giemsa staining) were purified from total peritoneal cells by a density gradient centrifugation with HistodenzTM
as previously described [31]. Since the density of Hdc−/− pMCs
was found to be lower than that of WT cells, 20% of HistodenzTM
solution was used.

Degranulation and granule protease assay
Degranulation of mast cells was evaluated by measuring enzyme
activity of a granule enzyme, β-hexosaminidase. Enzyme activityof β-hexosaminidase and three kinds of granule proteases were
measured as described previously [12]. Total β-hexosaminidase
activity in peritoneal cells and BMMCs was not significantly different between WT and Hdc−/− mice.

Oligonucleotide microarray
Total RNAs were labeled and hybridized to the MG U74AV2
murine GeneChip (Affymetrix, Santa Clara, CA) according to
the manufacturer’s instruction. We used the RMA (robust multiarray analysis) expression measure that represents the logtransform of (background corrected and normalized) intensities of the GeneChips. The RMA measures were computed
using the R package program, which is freely available on the
web site (http://www.bioconductor.org). We then removed all
genes whose maximum minus minimum values were less than 2(2-fold change), and selected 170 genes, which were differentially expressed between the wild and Hdc−/− mast cells. Using the
k-means clustering algorithm, these genes were classified into six
clusters on the basis of similarity of their expression profiles.

Quantitative RT-PCR analysis
Measurement of histamine
The amount of histamine was determined by the fluorometricalmethod with o-phthalaldehyde [32]. Cultured and pMCs weretwice washed in PBS and lysed in the PBS containing 0.5% Triton
X-100 and 2 M NaCl to obtain the soluble fraction for histamine
assay.

Flow cytometry
Flow cytometry analyses were performed with FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) equipped with the CELLQUEST
software as previously described.

C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Expression levels of granule proteases and histamine receptor
mRNAs in the cultured and pMCs were analyzed by quantitative RT-PCR with DNase-treated total RNAs. PCR was performed
using the specific primer pairs (forward, reverse) with Lightcycler
ST 300 (Roche Diagnostics, Basel, Switzerland); Hrh1 (H1 R), 5 TCA CTC CAG GCC TCA CAT GAC-3 , 5 -CAA AGT TCT CAT CCC
AAG TTT CCA-3 , HrH2 (H2 R), 5 -CAG TCC TAA GCG ACC CGG
TA-3 , 5 -GGC ACT GCT GGA TGT ATC TTG A-3 , HrH3 (H3 R), 5 ATG ACC GAT TCC TGT CAG TCA CTC-3 , 5 -TTC CGA ACA GCC
CGT CTT G-3 , HrH4 (H4 R), 5 -TGT GCA CCG CAT CTG TCT ACA
A-3 , 5 -GAA TCA TCG GGC CAT TTA CCA A-3 , Mcpt4 (MMCP-4),
5 -CCT TAC ATG GCC CAT CT-3 , 5 -CTT CCC CGG CTT GAT A-3 ,Mcpt5 (MMCP-5), 5 -AGA ACT ACC TGT CGG C-3 , 5 -GTC GTG
GAC AAC CAA AT-3 , Mcpt6 (MMCP-6), 5 -CTT TGA ACC GGA

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Eur. J. Immunol. 2014. 44: 204–214

TCG T-3 , 5 -CTC GTC ATT ATC AAT GTC GC-3 , Mcpt7 (MMCP7), 5 -AGC TAT GAC ACG AGA AGG-3 , 5 -GCT TAC GGA GCTGTA CT-3 , Ctsg, 5 -CTG TGT GGG AAA CCC GAG-3 , 5 -TCT TGG
TGC AAA GCG TCT-3 , and Gapdh, forward, 5 - TGT GTC CGTCGT GGA TCT GA-3 , reverse, 5 -TTG CTG TTG AAG TCG CAG
GAG-3 .

Innate immunity

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6 Tanaka, S., Nemoto, K., Yamamura, E. and Ichikawa, A., Intracellular
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Cytosolic Ca2+ concentration was measured as previously
described using Fura2-AM (Dojindo Laboratories, Kumamoto,Japan) [29]. Fluorescent intensities were measured, at an excitation wavelength of 340 or 380 nm and an emission wavelengthof 510 nm, with a fluorescence spectrometer (Jasco, CAF-100,
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7 Tanaka, S., Deai, K., Konomi, A., Takahashi, K., Yamane, H., Sugimoto,Y. and Ichikawa, A., Expression of L-histidine decarboxylase in granulesof elicited mouse polymorphonuclear leukocytes. Eur. J. Immunol. 2004.
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Reconstitution of mast cells in peritoneal cavity of themast cell deficient KitW /KitW-v mice

9 Ohtsu, H., Tanaka, S., Terui, T., Hori, Y., Makabe-Kobayashi, Y., Pejler,

BMMCs were injected into the peritoneal cavity (2 × 106
cells/cavity) of the 5-week-old male WBB6F1-KitW /KitW-v mice.
Peritoneal cells were collected 5 or 10 days after the engraftment
and then investigated.

10 Dy, M., Machavoine, F., Lebel, B., Ichikawa, A., Gastinel, L. N. andG., Tchougounova, E. et al., Mice lacking histidine decarboxylase exhibit
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Schneider, E., Interleukin 3 promotes histamine synthesis in hematopoietic progenitors by increasing histidine decarboxylase mRNA expression.
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stem cell factor. Proc. Natl. Acad. Sci. USA 1991. 88: 6382–6386.
12 Takano, H., Nakazawa, S., Okuno, Y., Shirata, N., Tsuchiya, S., Kainoh,

Data represent means ± SEM. Statistical significance for comparisons between groups was determined using Student’s paired t-testor ANOVA. Additional comparisons were made with Dunnett multiple comparison test for comparison with the control groups or
Tukey–Kramer multiple comparison test for all pairs of column
comparison. The values of “n” mean the number of the individual
trials with different animals.

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mast cells. FEBS Lett. 2008. 582: 1444–1450.
13 Schneider, E., Machavoine, F., Pleau, J. M., Berton, A. F., Thurmond, R.
L., Ohtsu, H., Watanabe, T. et al., Organic cation transporter 3 modulates
murine basophil functions by controlling intracellular histamine levels.
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14 Tanaka, S., Deai, K., Inagaki, M. and Ichikawa, A., Uptake of histamine
by mouse peritoneal macrophage cell line, RAW264.7. Am. J. Physiol. Cell
Physiol. 2003. 285: C592-C598.
15 Rabenstein, D. L., Bratt, P. and Peng, J., Quantitative characterization of
the binding of histamine by heparin. Biochemistry 1998. 37: 14121–14127.

Acknowledgments: This study was supported in part by a grantfrom Hyogo Science and Technology Association, Sankyo Foundation of Life Science, Takeda Science Foundation, and by JSPS
KAKENHI (grant numbers 19590091, 21790100, and 23590077).

16 Buckland, K. F., Williams, T. J. and Conroym, D. M., Histamine induces
cytoskeletal changes in human eosinophils via the H4 receptor. Br. J.
Pharmacol. 2003. 140: 1117–1127.
17 Lim, H. D., van Rijin, R. M., Ling, P., Bakker, R. A., Thurmond, R. L. andLeurs, R., Evaluation of histamine H1 -, H2 -, and H3 -receptor ligands at
the human histamine H4 receptor: identification of 4-methylhistamine
as the first and selective H4 receptor agonist. J. Pharmacol. Exp. Ther. 2005.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

314: 1310–1321.
18 Lim, H. D., Adami, M., Guaita, E., Werfel, T., Smits, R. A., de Esch, I.
J., Bakker, R. A. et al., Pharmacological characterization of the new histamine H4 receptor agonist VUF 8430. Br. J. Pharmacol. 2009. 157: 34–43.

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Full correspondence: Prof. Satoshi Tanaka, Department of
Immunobiology, Okayama University Graduate School of Medicine,
Dentistry, and Pharmaceutical Sciences, Okayama 700-8530, Japan
Fax: +81-86-251-7926
e-mail: tanaka@pharm.okayama-u.ac.jp
See accompanying Commentary:
http://dx.doi.org/10.1002/eji.201344262

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C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: 26/6/2013
Revised: 1/8/2013
Accepted: 29/8/2013
Accepted article online: 3/9/2013

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