International Immunology, Vol. 24, No. 2, pp. 107–116
Advance Access publication 9 January 2012
ª The Japanese Society for Immunology. 2012. All rights reserved.
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Histamine modulates multiple functional activities of
monocyte-derived dendritic cell subsets via
histamine receptor 2
Tu ¨nde Simon1,2, Pe ´ter Gogola ´k1, Katalin Kis-To ´th1, Ivett Jelinek2, Vale ´ria La ´szlo ´2and
E´va Rajnavo ¨lgyi1
1Department of Immunology, Medical and Health Science Center, University of Debrecen, Debrecen H-4032, Hungary
2Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest H-1089, Hungary
Correspondence to: E´. Rajnavo ¨lgyi, Institute of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen H-4032,
Nagyerdei Boulevard 98, Hungary; E-mail: email@example.com
Received 5 August 2011, accepted 22 November 2011
Expression of CD1a proteins in human monocyte-derived dendritic cells (DCs) specifies functionally
distinct subsets with different inflammatory properties. Histamine is recognized as an inflammatory
mediator released by various cell types including DCs. The diverse biological effects of histamine are
mediated by G-protein-coupled histamine receptors (HRs), which are able to modulate the functional
activities of DC subsets. The goal of the present study was to compare the expression and activity of
HRs in the CD1a2and CD1a1monocyte-derived DC subsets and to test the effects of histamine on the
differentiation, activation and functional activities of these subsets. We show that H2R is present at
high levels in both DC subsets, whereas H1R and H4R are expressed in a subset-specific manner.
Histamine shifts DC differentiation to the development of CD1a2DCs and modulates DC activation
through its inhibitory effect on CD1a1DC differentiation. Histamine-induced reduction of CD1a1DCs
is associated with increased secretion of IL-6 and IL-10, up-regulation of a typical combination of
chemokines, expression C5aR1 by the CD1a2DC subset and enhanced migration of both activated
DC subsets supported by the production of MMP-9 and MMP-12 enzymes. All these effects were
shown to be mediated in a H2R-specific manner as revealed by the specific antagonist of the receptor.
As H2R is expressed at high levels in both DC subsets, we propose that it may dominate the
regulation of multiple DC functions. In contrast, H1R and H4R with opposing subset-related
expression may have a regulatory or fine-tuning role in histamine-induced functional activities.
Keywords: CD1a molecule, cytokine, DC activation, migration
Dendritic cells (DCs) involve bone marrow-derived cells with
distinct life cycles, migratory properties and antigen present-
ing functions (1, 2). Being highly potent professional antigen-
presenting cells, DCs possess the unique potential to acti-
vate naive T cells, instruct Th-cell differentiation and thus
play a critical role in the induction and outcome of adaptive
immune responses (3, 4). CD34+hematopoietic progenitors
are widely studied precursors of DCs that give rise to
several classical DC subsets when cultured in the presence
of appropriate cytokine combinations (5, 6). DCs can also
differentiate from CD14+blood monocytes in peripheral tis-
sues and lymphoid organs or under in vitro conditions
(7, 8). Recent studies identified several subsets of conven-
tional circulating DCs with unique phenotypic and functional
properties both in mouse and human (9–12) demonstrating
the functional specialization of these cells.
Originally, in vitro generated human monocyte-derived
DCs have been considered as a homogeneous and func-
tionally competent cell population appropriate for clinical util-
ity (13). However, the expression pattern of human CD1
membrane proteins has been shown to specify distinct hu-
man DC subtypes independent on their activation state
(14, 15). We have previously shown that human monocyte-
derived DCs generated in vitro involve both CD1a?and
CD1a+cells, their ratio varies among individuals and is con-
trolled by the lipid and lipoprotein environment of DCs via
the ligand-activated nuclear hormone receptor peroxisome
proliferator-activated receptor gamma (16). It has also been
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demonstrated that CD1a?and CD1a+DCs exhibit distinct
functional characteristics being the CD1a+subset more
inflammatory as compared with its CD1a?counterpart (8).
One of the major functions of DCs is the continuous sam-
pling of their environment by receptors expressed in the cell
or vesicular membranes or in the cytosol and transfers this
information to T lymphocytes (17). Some of these receptors
mediate internalization of soluble material or particles; others
are involved in signal transduction, cell migration or cell-to-
cell communication (18). As a result, the differentiation and
activation of DCs occur in an environment-dependent man-
ner and result in considerable functional flexibility. Depend-
ing on the actual tissue environment, phenotypically defined
monocyte subsets develop to monocyte-derived DCs, which
also represent a functionally heterogeneous cell population
(11). Under in vivo conditions, DC differentiation and activa-
tion depend on the modulatory effects of metabolites, cyto-
kines,chemokines and other
a multifunctional small-sized biogenic amine that regulates
cellular responses and plays diverse roles in physiological
and pathological processes (19, 20). Histamine was also
shown to influence DC differentiation, as both the intracellu-
lar histamine content and the expression of the histidine
decarboxylase enzyme were found to be increased in the
course of in vitro cytokine-induced monocyte-derived DC dif-
ferentiation in parallel with the induction of the co-stimulatory
molecules CD40, CD80 and CD86 (21).
A rapidly growing body of evidence highlighted that hista-
mine, a small biogenic amine, is involved in the regulation
of DC functions and consequently T cell-mediated inflamma-
tion (22). Histamine is recognized as an inflammatory media-
tor released by mast cells, basophils and other cell types,
including DCs themselves (21). The diverse biological
effects of histamine are mediated via four distinct G-protein-
coupled receptors, namely H1, H2, H3 and the lastly discov-
ered H4 receptors (23). It is well documented that H1, H2
and H4 receptor mRNAs and proteins are expressed in hu-
man DCs (24–27) but the presence of H3R is still controver-
sial (28, 29). Interestingly, neither H1R nor H2R could be
detected on the surface of Langerhans cells (LCs) isolated
from human epidermis or differentiated in vitro (30). In con-
trast to these results, the expression of the recently discov-
ered H4R was detected at both mRNA and protein levels on
primary LCs of both murine and human skin samples and
also on in vitro generated monocyte-derived LCs (31, 32).
The expression pattern and functionality of histamine recep-
tors (HRs) in various DC subpopulations, however, are
The majority of the experimental data support that hista-
mine induces an altered cytokine expression profile in DCs
and favor Th2 polarization. Histamine was also shown to in-
hibit the secretion of the pro-inflammatory cytokines IL-1b
and IL-6 as well as the Th1 polarizing cytokine IL-12p70 (24).
These effects could be antagonized by specific HR antago-
nists (24, 25, 33), whereas the production of Th2 cytokines
and LPS-induced IL-8 and IL-10 production was increased in
histamine-treated human monocyte-derived DCs (28, 33). The
reduced production of the Th1 polarizing cytokine IL-12p70
was mediated not only by H2R but also by H4R (29). Several
independent research groups also established that histamine
(26, 29) or histamine agonists (28, 29) act as strong chemo-
taxins for resting but not for activated DCs. The involvement
of H1R (28), H2R (34, 35), H3R (26, 28) and H4R (36, 37) has
been shown to exert modulatory effects on DC migration.
The major goal of the present study was to compare the
expression of HRs in the previously identified and character-
ized CD1a?and CD1a+human monocyte-derived DC sub-
populations and to test the modulatory effects of histamine
on the differentiation, activation, cytokine and chemokine se-
cretion and migration of these DC subsets. Specific inhibi-
tors of the H1, H2 and H4 receptors were used to validate
the functionally relevant effects on DC activities and to iden-
tify the HRs responsible for the DC-associated modulatory
effects of histamine.
Monocyte separation and differentiation of DCs
Buffy coats were obtained with the written permission of the
National Blood Transfusion Centre, Budapest Hungary. Pe-
ripheral blood mononuclear cells (PBMC) were obtained by
Uppsala, Sweden). Monocytes were isolated from PBMC by
microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany).
CD14highmonocytes (>97%) were cultured in 6-well plates at
a density of 2 3 106cells ml?1in serum-free AIMV medium
(Life technologies, Carlsbad, CA, USA) supplemented with
antibiotics, 100 ng ml?1IL-4 and 80 ng ml?1GM-CSF. At day
2, the same amount of cytokines was added and the cells
were cultured for another 3 days. Resting DCs were har-
vested on day 5 or were activated by LPS (100 ng ml?1,
Sigma–Aldrich, Schnelldorf, Germany) and IFNc (10 ng ml?1,
PeproTech Inc., Rocky Hill, NJ, USA) and harvested on day
6. When indicated, histamine (10?5M, Sigma–Aldrich) or his-
tamine combined with specific HR inhibitors (10?5M) (38,
39) were added at days 0 and 2 together with the differentiat-
ing cytokines and when activated, with LPS+IFNc (100 ng ml?1
and 10 ng ml?1, respectively). The following HR antagonists
were used: pyrilamine (H1R antagonist, Sigma–Aldrich),
famotidine (H2R antagonist Sigma–Aldrich), JNJ7777120 and
JNJ10191584 (H4R antagonists, Sigma–Aldrich and Tocris
Bioscience, Ellisville, MO, USA).
Phenotyping of resting and activated DCs was performed by
flow cytometry using anti-CD83-FITC and anti-CD1a-PE anti-
bodies (Beckman Coulter, Hialeah, FL, USA) and isotype-
matched control antibody (BD Pharmingen, San Diego, CA,
USA). For measuring H2R expression by DC subsets, the
washed cells were labeled with 0.1 lg rabbit anti-human
H2R (Alpha Diagnostic International Inc., San Antonio, TX,
USA) in the presence of heat-inactivated normal mouse se-
rum as a blocking reagent. After 30 min incubation on ice,
the cells were washed and labeled with 1 lg (0.5 ll)
Alexa488-conjugated goat anti-rabbit (Fab’)2fragments (Invi-
trogen, Eugene, OR, USA) together with 5 lPE-labeled
mouse anti-human CD1a antibody (BD Biosciences, Franklin
Lakes, NJ, USA). Fluorescence intensities were measured
108 H2R modulates monocyte-derived DC differentiation
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by FACS Calibur (BD Biosciences); data were analyzed by
the FlowJo software (Tree Star, Ashland, OR, USA). The
CD1a?and CD1a+cells were separated with FACS DiVa
high-speed cell sorter (BD Biosciences).
Real-time quantitative reverse transcriptase-PCR
Total RNA was isolated from DCs by Trizol reagent (Invitro-
gen, Carlsbad, CA, USA). Reverse transcription was per-
formed at 37?C for 120 min from 100 ng total RNA using the
High Capacity cDNA Archive Kit (Applied Biosystems, Fos-
ter City, CA). Quantitative reverse transcriptase (QRT)-PCR
for the HRs H1, H2, H3, H4, MMP-9 and MMP-12 genes
was performed by ABI PRISM 7900 (Applied Biosystems)
with 40 cycles at 94?C for 12 s and 60?C for 60 s using Taq-
man gene expression assays (Applied Biosystems). All PCR
reactions were run in triplicates with a control reaction con-
taining no RT enzyme. The comparative Ct method was used
to quantify transcripts relative to the endogenous control
genes GAPDH or 36B4.
Culture supernatants of LPS+IFNc activated DCs were har-
vested 24 h after LPS activation and the concentration of IL-
6 and IL-10 was measured by using OptEIA kits (BD
DCs were suspended in migration medium (0.5% BSA in
RPMI 1640) at 106cells ml?1. Transmigration inserts with 6.5
mm diameter and 5 lm pore size were obtained from BD
Biosciences. The MIP-3b chemokine (PeproTech) was di-
luted at 200 ng ml?1in migration medium and added to the
lower chambers in a final volume of 600 ll. DCs were added
to the upper chamber in a final volume of 250 ll and the
chemotaxis assay was conducted for 4 h in 5% CO2 at
37?C. At the end of the assay, the inserts were discarded
and cells migrated to the lower chamber were collected.
The number of cells migrated was counted by using polysty-
rene standard beads (Sigma–Aldrich) by flow cytometry.
Chemokine gene expression profiling of CD1a?and CD1a+
DCs was studied with the Human Chemokines & Receptors
PCR Array assay (SABiosciencesTM, Frederick, MD, USA) in
accordance with the manufacturer’s recommendations. Briefly,
total RNA was isolated from the different cell populations using
RNeasy Mini Kit (Qiagen, Duesseldorf, Germany). After DNase
I (Qiagen) digestion, first strand cDNA synthesis was per-
formed by using the RT2First Strand Kit (SABiosciencesTM)
following the manufacturer’s instructions and including a geno-
mic DNA elimination step and external RNA controls. For each
sample, 900 ng of total RNA was reverse transcribed. Real-
time PCR measurement was performed using the RT2qPCR
Master mix (SABiosciencesTM) according to the manufac-
turer’s instructions on the ABI Prism 7000 real-time PCR plat-
form. For each analysis, 25 ll of the experimental cocktail of
the cDNA samples and RT2qPCR Master mix was distrib-
uted across the PCR array 96-well plates, each of which
contained 84 wells with a real-time PCR assay for different
chemokine genes, 5 wells with assays for different house-
keeping genes, a genomic DNA control, 3 replicate reverse
transcription controls and 3 replicate positive PCR controls.
A two-step cycling program was used consisting of an initial
activating step of 10 min at 95?C and a second step of
40 cycles of denaturation (95?C, 15 s) and annealing (60?C,
1 min). Finally, each plate underwent a melting curve
program [95?C, 1 min; 65?C, 2 min (optics off); 65–95?C at
(optics on)]. Gene expression levels were
determined as the inverse of the Ct value. Ct values greater
than 35, or those not detected, were set to 35. After cycling
with real-time PCR, the amplification data were analyzed and
statistical significance was calculated by SABiosciences online
software. Cytokines, chemokines and their receptors genes not
modulated by histamine are listed in Supplementary Figure 1,
available at International Immunology Online.
Histamine modulates monocyte-derived DC differentiation
First, we studied how histamine, potentially acting through
different HRs, can modulate the differentiation of the previously
identified CD1a?and CD1a+DC subsets. By using IL-4
and GM-CSF, we generated monocyte-derived DCs in the
presence or absence of histamine and found that histamine
significantly reduced the proportion of CD1a+DCs (Fig. 1A,
black bar) as compared with cells generated in the absence
of histamine. To identify the HR involved in this effect, we
used specific pharmacological blocking agents and showed
that the presence of the H2R antagonist famotidine could
prevent the reducing effect of histamine on CD1a+cell differ-
entiation (Fig. 1A and B). The H1R and the H4R antagonists
had no or only marginal effects (Fig. 1A). Flow cytometric
analysis showing the cell surface expression of H2R on
CD1a?and CD1a+DC subsets confirmed the inhibitory ef-
fect of histamine on CD1a+cell differentiation at the protein
level and also the efficient blocking of this effect by famoti-
dine, but not by the other inhibitors (Fig. 1B). These results
show that histamine exerts a DC subset-specific effect on
monocyte to DC differentiation. As famotidine was the only in-
hibitor that could restore CD1a+DC generation, whereas the
JNJ7777120 and JNJ10191584 had no such effect, these
results suggest that H2R is preferentially responsible for this
and the H4Rblockers
Expression of HRs in CD1a?and CD1a+DC subsets
The expression of HRs by human DCs has previously been
demonstrated (24–29); however, their expression in CD1a?
and CD1a+DC subsets has not been investigated so far. To
assess the DC subset-specific expression of HRs in in vitro
differentiated monocyte-derived DCs obtained from nine
healthy individuals with an average of 46 6 11% CD1a+ra-
tios, we sorted the cells to CD1a?and CD1a+subpopula-
tions by flow cytometry and compared the expression of
H1R, H2R and H4R measured by QRT-PCR. H3R expression
could not be detected; H1R expression was significantly
higher in CD1a?DCs, H4R was higher in CD1a+cells as
H2R modulates monocyte-derived DC differentiation 109
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compared with their CD1a?counterpart, whereas H2R was
expressed by both subsets at high levels (Fig. 2A). This find-
ing was also supported by the detection of H2R by flow
cytometric analysis on the surface of both CD1a?and
CD1a+cells at comparable levels (Fig. 2B). These results
revealed the subset-specific expression of H1R and H4R
and identified H2R as the dominant HR in human monocyte-
derived DCs irrespective of their subset.
Effect of histamine on the activation of CD1a?and CD1a+DC
Previously, we described that inflammatory cytokines can
block the transition of CD1a?cells to CD1a+DCs and thus
stabilize the CD1a?phenotype for further activation (8). To
monitor the effect of histamine on the activation of CD1a?
and CD1a+subsets, DCs were generated in the presence
of histamine and activated by the combination of LPS and
IFNc. Cell surface expression of CD1a and the activation
marker CD83 were monitored in the CD1a?and CD1a+DC
subsets by flow cytometry. The results summarized in
Fig. 3A–C show that histamine did not effect significantly the
expression of CD83, and both the CD1a?and CD1a+sub-
sets could be activated by LPS+IFNc. Histamine, present in
the course of in vitro monocyte-derived DC differentiation,
was able to reduce the proportion of CD1a+cells (Fig. 1)
and consequently decreased the ratio of both resting and
activated CD1a+CD83+cells, while the CD1a?CD83+fraction
became larger only in mature DCs (Fig. 3A–C). In line with
our results obtained with resting DCs, the H2R antagonist
Fig. 1. The effect of histamine and pharmacological HR antagonists on
the development of CD1a?and CD1a+monocyte-derived dendritic cell
subsets. The percentage of CD1a+cells was detected by flow
cytometry in in vitro generated resting monocyte-derived DCs, in DCs
differentiated in the presence of histamine and in DCs differentiated in
the presence of histamine in combination with the H1R antagonist
pyrilamine, the H2R antagonist famotidine or the H4R antagonists
JNJ7777120 and JNJ10191584 (A). Mean 6 SD of three independent
experiments are presented. The histograms show the distribution of
CD1a?and CD1a+cells generated in the presence or absence of
histamine or in the presence of histamine and famotidine (B).
Fig. 2. Gene profiling of histamine receptors in monocyte-derived
dendritic cell subsets. DCs were generated with or without histamine
added to the culture medium together with the differentiating cytokines
IL-4 and GM-CSF on days 0 and 2 of in vitro cultures. On day 5, the
cells were stimulated by LPS+IFNc for 24 h. A fraction of cells was left
untreated for using it as a non-activated DC control. Resting and
activated DCs were harvested on days 5 and 6, respectively, and
subjected to RNA isolation and reverse transcription to prepare cDNA.
Gene expression of H1R, H2R and H4R was measured in DCs isolated
from eight different donors, all sorted to CD1a?and CD1a+DC
fractions (A). QRT-PCR was performed in triplicates and the compar-
ative Ct method was used to quantify transcripts relative to the
endogenous housekeeping gene GAPDH. Expression of H2R was
detected by flow cytometry using indirect staining (B).
110H2R modulates monocyte-derived DC differentiation
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famotidine suspended this histamine-mediated effect (Fig.
3A–C), while the H1R antagonist pyrilamine further reduced
the ratio of CD1a+CD83+cells. These results suggest that
histamine modulates DC activation through its inhibitory
effect on CD1a+DC differentiation.
DCs can be activated by various stimuli, which result in
the secretion of cytokines and chemokines (40). Our previ-
ous results demonstrated that upon activation by CD40L or
TLR ligands, the CD1a?and CD1a+DCs secrete different
sets of cytokines (8). To test whether histamine- or HR-
specific antagonists can modify the cytokine production of
DC subsets, we measured the concentrations of cytokines
in the culture supernatant of activated CD1a?and CD1a+
DC cultures by ELISA. Statistically significant increase of the
secreted pro-inflammatory cytokine IL-6 (Fig. 3D) and the
regulatory cytokine IL-10 (Fig. 3E) could be shown when
DCs were generated in the presence of histamine. This ef-
fect also could be inhibited by famotidine indicating again
the involvement of H2R in this process. The secretion of IL-
12p70 was detected preferentially in DCs with high, >60%
of CD1a+cell content and its production could not be con-
sistently inhibited by famotidine (data not shown). These
results demonstrate that the presence of histamine results in
a decreased proportion of the CD1a+DC subset and this has
a significant impact on the production of IL-6 and IL-10 cyto-
kines. As CD1a+DCs have previously been identified as po-
tent producers of IL-12p70, we propose that the increase of
IL-10 secretion can at least partially be the consequence of
altered cytokine balance in the DC microenvironment.
Effect of histamine on DC migration
Histamine not only induces cell migration mediated by differ-
ent HRs (26, 28, 29), but at the same time, it is also able to
stimulate matrix degrading enzyme production (41). Migration
of DCs in blood and lymph and from peripheral tissues to
Fig. 3. Effect of histamine on the induction of monocyte-derived dendritic cell activation. DCs were differentiated in the presence of histamine as
described in Fig. 1 and the differentiated cells were activated by LPS+IFNc on day 5 of culture. Cells were harvested on days 5 and 6 and the
expression of CD83 was measured on the surface of immature (IDC) and LPS+IFNc-activated CD1a?and CD1a+cells (A–C). F and P refer to
famotidine and pyrilamine, respectively. The histamine-modulated cytokine secretion by activated DCs was monitored by measuring the
concentrations of IL-6 (D) and IL-10 (E) cytokines in the culture supernatants of resting and activated DCs differentiated in the absence or
presence of histamine or histamine together with the H2R antagonist famotidine. Mean 6 SD of triplicate measurements performed with DCs of
six independent experiments are shown.
H2R modulates monocyte-derived DC differentiation 111
by guest on November 24, 2015
secondary lymphoid organs is regulated at multiple levels
(42, 43). Our in vitro migration experiments revealed that the
presence of histamine during monocyte-derived DC differenti-
ation significantly enhanced the migratory potential of DCs
that could be shown at the level of both spontaneous and
MIP-3b-induced migration (Fig. 4A). Famotidine, the specific
H2R antagonist, was able to reverse this effect (Fig. 4B)
pointing to the role of H2R in the modulation of cell mobility.
Monitoring mRNA expression of MMP-9 and MMP-12 showed
that the presence of histamine was able to up-regulate the
mRNA expression of these matrix degrading enzymes in both
resting and activated DCs (Fig. 4C and D). This effect could
also be reversed by famotidine (Fig. 4C and D) further
supporting the role of H2R in regulating DC migration.
Histamine-induced expression of C5a receptor is restricted to
the CD1a?DC subset
In the next set of experiments, we performed a global screen-
ing using a Q-PCR based array to identify chemokines and
chemokine receptors involved in the histamine-induced H2R-
mediated enhancement of DC migration. Following LPS+IFNc
activation, the monocyte-derived DCs generated in the pres-
ence of histamine, famotidine or histamine together with famo-
tidine were separated to CD1a?and CD1a+subsets and their
chemokine and chemokine receptor expression profiles were
determined. When DCs were generated in the presence of his-
tamine, increased mRNA expression of C5aR1 was detected
in the CD1a+subset and famotidine abolished this effect in
this subset (Fig. 5A). However, C5aR1 protein expression
clearly showed that its expression was dramatically up-
regulated by histamine preferentially in the CD1a?subset
at both their resting (Fig. 5B) and activated (Fig. 5C) differ-
entiation states. As famotidine could completely abolish the
effect of histamine, these results also revealed the H2R de-
pendency of this histamine-induced effect.
In addition to identifying C5aR1 as a histamine-induced
chemotactic receptor of DCs preferentially expressed by the
CD1a?subset, we also identified further migration-related
Fig. 4. The effect of histamine- on monocyte-derived dendritic cell migration. Migration of activated DCs generated with or without histamine was
measured in the absence or presence of MIP-3b chemokine by the transwell system (A and B). The effects of histamine on the expression of
matrix metalloproteinases MMP-9 and MMP-12 were determined by measuring relative mRNA expression levels by QRT-PCR (C and D). IDC and
MDC indicate immature and mature DCs, respectively. Mean 6 SD of five independent experiments are shown.
112H2R modulates monocyte-derived DC differentiation
by guest on November 24, 2015
genes as summarized in Table 1. The expression of CXCR4
and CX3CR1 also increased significantly in DCs generated in
the presence of histamine and this effect on CX3CR1 could
be inhibited by famotidine in the CD1a+DC subset, while in
CD1a?cells histamine did not exert this modulatory effect.
In this study, we investigated the effects of histamine on the
differentiation and activities of two, developmentally related
and in vivo relevant monocyte-derived DC subsets gener-
ated from CD14highmonocytes (8). We found that (i) hista-
development of CD1a?DCs; (ii) this effect was attributed to
H2R as only its specific synthetic inhibitor could restore the
generation of CD1a+cells; (iii) H2R was highly expressed
by both DC subsets, whereas H1R and H4R were expressed
in a subset-specific manner; (iv) histamine modulated DC
activation through its inhibitory effect on CD1a+DC differen-
tiation; (v) histamine-induced reduction of CD1a+
resulted in increased secretion of IL-6 and IL-10; (vi) hista-
mine modulated the expression of C5aR1 in a H2R- and
subset-specific manner; (vii) histamine induced spontane-
ous- and chemokine-mediated DC migration of both subsets
and modulated the production of the MMP-9 and MMP-12
enzymes also involved in the regulation of DC migration.
DC differentiation is a complex and highly regulated pro-
cess driven by the actual tissue environment of the cell. Lip-
ids and lipoproteins have previously been identified as
modulators of the cell surface expression of type I and type
II CD1 proteins, which act as both presenting molecules of
microbial glyco- and phopholipids and also as phenotypic
markers of human DC subsets (44). The modulatory effect of
histamine on the expression of CD1a and the in vitro differen-
tiation of DCs has previously been reported (45). Here, we
not only confirmed these findings but extended our studies to
demonstrate that histamine has an impact on other DC func-
tions which may be regulated in a subset-specific manner.
DC functions may be associated to more than one HR,
thus we sought to generate monocyte-derived DCs in vitro
in the presence of histamine or histamine in combination
with pharmacological antagonists of H1R, H2R and H4R. As
famotidine, a potent antagonist of H2R was the only com-
pound that could prevent the histamine-mediated inhibition
of CD1a+DC differentiation we identified H2R as the recep-
tor involved in this regulation. Several research groups dem-
onstrated the expression HRs in human DCs (24–29);
Fig. 5. The effect of histamine on the expression of C5aR1 gene and protein. Resting and activated monocyte-derived DCs, differentiated in the
absence or presence of histamine or in the presence of histamine in combination of famotidine, were sorted to CD1a?and CD1a+DC subsets.
The subset-specific expression of C5aR1 in the CD1a?and CD1a+DCs was determined at both mRNA (A) and protein (B and C) levels
measured by a Q-PCR array and by flow cytometry, respectively. Mean 6 SD of three independent experiments are shown.
H2R modulates monocyte-derived DC differentiation113
by guest on November 24, 2015
however, their expression in CD1a?and CD1a+cells has not
been analyzed so far. Similar to previous results (46), we
could not detect H3R expression in DCs but found high
expression of H2R in both CD1a?and CD1a+DCs. Interest-
ingly, H1R and H4R mRNA expression showed a subset-
dependent pattern: CD1a?
higher levels of H1R than CD1a+cells, whereas H4R expres-
sion was significantly higher in the CD1a+DC subset. This
finding raises interesting questions concerning the role of
H4R in a situation where due to the presence of histamine
the generation of the cell type i.e. CD1a+DCs carrying the
receptor at high levels is inhibited. Based on this scenario
and taken the previously described inflammatory, nature of
CD1a+DCs in individuals with high CD1a+DC ratios may
develop more severe inflammation in the presence of hista-
mine than those with low CD1a+numbers. Alternatively, this
expression pattern may have a regulatory function to keep
histamine-mediated regulation under check.
DC maturation is initiated by the engagement of different
surface receptors and results in phenotypic and functional
changes. Histamine was shown to induce transient up-
regulation of MHC class II proteins and the co-stimulatory
molecules CD40, CD80 and CD86 in human monocyte-
derived DCs and antagonists of both H1R and H2R pre-
vented histamine-induced CD86 expression (24) but did not
modified CD40 expression (33). When DCs were activated
with LPS+IFNc, we found that due to the histamine-induced
reduction of CD1a+DC numbers, the ratio of CD1a+CD83+
cells decreased, while the CD1a?CD83+cell fraction be-
came more prominent. This observation demonstrates that
histamine, by counteracting CD1a+DC differentiation, has
an impact on the DC subset distribution of mature DCs in
a H2R-dependent manner.
The hallmark of activated DCs is the production of cyto-
kines and chemokines, which may act in an autocrine or para-
DCs expressed significantly
crine manner. Previous results demonstrated that histamine
induced altered cytokine production in DCs, supported polari-
zation of T lymphocytes to Th2 direction (24), and the cytokine
profile of resting CD1a?and CD1a+DCs was different (47). In
line with these findings, we also observed that upon activation
by CD40L or TLR ligands, both CD1a?and CD1a+DCs se-
creted pro-inflammatory cytokines but IL-12p70 and CCL1
production was mainly attributed to the CD1a+, whereas IL-
10 secretion to the CD1a?subset (8). DCs generated in the
presence of histamine and activated by LPS+IFNc produced
significantly higher levels of the pro-inflammatory cytokine IL-
6 and the regulatory cytokine IL-10 as compared with cells
generated in the absence of histamine. Again, famotidine
was the only inhibitor that could interfere with this effect. In
our studies, IL-12p70 secretion was detected in DCs with
high (>60%) CD1a+cell content and famotidine had no signif-
icant effect on cytokine secretion indicating that IL-12p70 se-
cretion is not dependent on H2R. In a previous study, IFNc
was shown to up-regulate H4R and its stimulation resulted in
the down-regulation of IL-12p70 and CCL2 production in
human monocyte-derived DCs (31).
Resting DCs migrate to both body surfaces and interstitial
spaces where they encounter with self- or foreign antigens.
After antigen challenge, migration of activated DCs through
lymphatics ensures DC T-cell contact in lymphoid organs.
Several independent research groups found that histamine
(26, 29) or histamine agonists (28, 29) exerted chemotactic
effects for resting, but not for activated DCs (28). By using
specific receptor antagonists, the involvement of both H3R
(26, 28) and H1R (28) has been demonstrated as modula-
tors of cell migration. In a skin DC migration assay, both his-
tamine and a H4R agonist induced enhanced chemotaxis,
which could be blocked by both H1R and H4R antagonists.
Similar results were obtained in vitro by using bone marrow-
derived mouse DCs (36). Our previous results also verified
the role of H4R in murine DC migration (48).
In our present migration experiments, histamine increased
both spontaneous- and MIP-3b-induced migration of DCs.
Famotidine was the only HR antagonist that could reverse
this effect. Histamine was also shown to stimulate the pro-
duction of the matrix degrading enzymes and the histamine-
induced increase of MMP-13 and MMP-3 production was
shown in vitro (41). The essential role of MMP-9 in DC migra-
tion was measured in reconstituted basement membrane
(Matrigel) and in vivo by migration to the draining lymph node
(49) and the expression of MMP-12 in DCs was also shown
(50). Histamine induced up-regulation of MMP-9 and MMP-12
and their inhibition by the H2R blocker famotidine confirmed
the dominant role of H2R in this DC function as well.
Migration of DCs in blood and lymph and from peripheral
tissues to secondary lymphoid organs is regulated at multi-
ple levels (42, 43) and a number of cytokines, chemokines
and their receptors are implicated in the regulation of this
complex process. The combination of chemokine receptors,
induced in activated DCs when developed in the presence
of histamine and stimulated with LPS+IFNc involved C5aR1,
CXCR4 and CX3CR1, previously identified in the sulpho-Lac-
NAc expressing DCs (slanDCs/MDC-8), which represent the
largest population of blood DCs (51). These circulating DCs
are highly pro-inflammatory due to their production of TNF-a
Table 1. Relative expression of chemokine receptor genes
modulated by histamine in monocyte-derived dendritic cell
subsets through H2R. CD1a?and CD1a+monocyte-derived
DCs were differentiated and treated as described in Fig. 5.
Comparison of the differentially treated DC samples was
expressed as fold increase of mRNA levels in CD1a+versus
Gene Compared groupsFold changeP value
Activated CD1a?/CD1a+DC 2.92
Differentiated in the
Differentiated in the
histamine + famotidine
Differentiated in the
presence of histamine +
Statistically significant results are indicated in bold.
114H2R modulates monocyte-derived DC differentiation
by guest on November 24, 2015
and IL-12p70, but in contrast to CD1c+conventional blood
DCs, they are negative for the CD11c, CD14 and CD1
markers. C5a is a pro-inflammatory mediator that has re-
cently been detected in monocyte-derived DCs (52) and sig-
naling through C5aR1 has been found to regulate the
development of Treg and Th17 cells (53). Here, we show for
the first time that monocyte-derived DCs, differentiated in
the presence of histamine and activated by LPS+IFNc, pref-
erentially give rise to CD1a?C5aR1+cells.
CXCR4 endowed with potent chemotactic activity is up-
regulated upon DC activation induced by LPS, TNF-a or
CD40L (54) and engagement by its specific ligand CXCL12/
stromal-cell derived factor-1 (SDF-1a) promotes DC activa-
tion, survival and chemotaxis both in vitro and in vivo (55).
The CX3CR1 transmembrane protein has been suggested
to be enhanced by IFNc in TNF/iNOS-producing DCs differ-
entiated from classical monocytes (56) and it is involved in
the recruitment of DCs by its specific ligand CX3CL1/fractal-
kine (57). Based on these results, we propose that hista-
mine, in combination with inflammatory signals, is able to
induce the expression of a typical combination of chemokine
receptors that also modulate monocyte-derived DC func-
tions. However, this effect can be further up-regulated in the
presence of the CCL19/MIP-3b chemokine, which specifi-
cally attracts CCR7 expressing activated DCs.
In summary, our results demonstrate that histamine has
a profound effect on the development of CD1a+DCs that is
mediated by the H2R. This regulation has a further impact on
cytokine production known to be different in the CD1a?and
CD1a+subsets. As H2R is expressed at high levels in both
DC subsets, it may dominate the regulation of multiple DC-
related functional activities. In contrast, H1R and H4R with op-
posing subset-related expression may have a regulatory or
fine-tuning role in histamine-induced functional activities.
Supplementary data are available at International Immunology
National Science Foundation (OTKA NK-72937); Research
University (TA´MOP-4.2.1/B-09/1/KONV-2010-0007); Innova-
The excellent technical assistance of Erzse ´bet Nagy is appreciated.
1 Banchereau, J., Briere, F., Caux, C. et al. 2000. Immunobiology of
dendritic cells. Annu. Rev. Immunol. 18:767.
2 Merad, M. and Manz, M. G. 2009. Dendritic cell homeostasis.
3 Schuurhuis, D. H., Fu, N., Ossendorp, F. and Melief, C. J. 2006. Ins
and outs of dendritic cells. Int. Arch. Allergy Immunol. 140:53.
4 McCullough, K. C. and Summerfield, A. 2005. Basic concepts of
immune response and defense development. ILAR J. 46:230.
5 Caux, C., Vanbervliet, B., Massacrier, C. et al. 1996. CD34+
hematopoietic progenitors from human cord blood differentiate
along two independent dendritic cell pathways in response to GM-
CSF+TNF alpha. J. Exp. Med. 184:695.
6 Gatti, E., Velleca, M. A., Biedermann, B. C. et al. 2000. Large-
scale culture and selective maturation of human Langerhans cells
from granulocyte colony-stimulating factor-mobilized CD34+ pro-
genitors. J. Immunol. 164:3600.
7 Cheong, C., Matos, I., Choi, J. H. et al. 2010. Microbial stimulation
fully differentiates monocytes to DC-SIGN/CD209(+) dendritic
cells for immune T cell areas. Cell 143:416.
8 Gogolak, P., Rethi, B., Szatmari, I. et al. 2007. Differentiation of
CD1a- and CD1a+ monocyte-derived dendritic cells is biased by
lipid environment and PPARgamma. Blood 109:643.
9 Shortman, K. and Naik, S. H. 2007. Steady-state and inflammatory
dendritic-cell development. Nat. Rev. Immunol. 7:19.
10 Helft, J., Ginhoux, F., Bogunovic, M. and Merad, M. 2010. Origin
and functional heterogeneity of non-lymphoid tissue dendritic
cells in mice. Immunol. Rev. 234:55.
11 Geissmann, F., Manz, M. G., Jung, S., Sieweke, M. H., Merad, M.
and Ley, K. 2010. Development of monocytes, macrophages, and
dendritic cells. Science 327:656.
12 Bachem, A., Guttler, S., Hartung, E. et al. 2010. Superior antigen
cross-presentation and XCR1
CD11c+CD141+ cells as homologues of mouse CD8+ dendritic
cells. J. Exp. Med. 207:1273.
13 Romani, N., Reider, D., Heuer, M. et al. 1996. Generation of mature
dendritic cells from human blood. An improved method with
special regard to clinical applicability. J. Immunol. Methods
14 Nestle, F. O., Zheng, X. G., Thompson, C. B., Turka, L. A. and
Nickoloff, B. J. 1993. Characterization of dermal dendritic cells
obtained from normal human skin reveals phenotypic and
functionally distinctive subsets. J. Immunol. 151:6535.
15 Larregina, A. T., Morelli, A. E., Spencer, L. A. et al. 2001. Dermal-
resident CD14+ cells differentiate into Langerhans cells. Nat.
16 Szatmari, I., Gogolak, P., Im, J. S., Dezso, B., Rajnavolgyi, E. and
Nagy, L. 2004. Activation of PPARgamma specifies a dendritic cell
subtype capable of enhanced induction of iNKT cell expansion.
17 Cahalan, M. D. and Gutman, G. A. 2006. The sense of place in the
immune system. Nat. Immunol. 7:329.
18 Iwasaki, A. and Medzhitov, R. 2010. Regulation of adaptive
immunity by the innate immune system. Science 327:291.
19 Jones, B. L. and Kearns, G. L. 2011. Histamine: new thoughts
about a familiar mediator. Clin. Pharmacol. Ther. 89:189.
20 Akdis, C. A., Jutel, M. and Akdis, M. 2008. Regulatory effects of
histamine and histamine receptor expression in human allergic
immune responses. Chem. Immunol. Allergy 94:67.
21 Szeberenyi, J. B., Pallinger, E., Zsinko, M. et al. 2001. Inhibition of
effects of endogenously synthesized histamine disturbs in vitro
human dendritic cell differentiation. Immunol. Lett. 76:175.
22 Vanbervliet, B., Akdis, M., Vocanson, M. et al. 2011. Histamine
receptor H1 signaling on dendritic cells plays a key role in the
IFN-gamma/IL-17 balance in T cell-mediated skin inflammation.
J. Allergy Clin. Immunol. 127:943.
23 Jutel, M., Blaser, K. and Akdis, C. A. 2006. The role of histamine in
regulation of immune responses. Chem. Immunol. Allergy 91:174.
24 Caron, G., Delneste, Y., Roelandts, E. et al. 2001. Histamine
induces CD86 expression and chemokine production by human
immature dendritic cells. J. Immunol. 166:6000.
25 Gutzmer, R., Langer, K., Lisewski, M. et al. 2002. Expression and
function of histamine receptors 1 and 2 on human monocyte-
derived dendritic cells. J. Allergy Clin. Immunol. 109:524.
26 Damaj, B. B., Becerra, C. B., Esber, H. J., Wen, Y. and
Maghazachi, A. A. 2007. Functional expression of H4 histamine
receptor in human natural killer cells, monocytes, and dendritic
cells. J. Immunol. 179:7907.
27 Amaral, M. M., Davio, C., Ceballos, A. et al. 2007. Histamine
improves antigen uptake and cross-presentation by dendritic
cells. J. Immunol. 179:3425.
28 Idzko, M., la Sala, A., Ferrari, D. et al. 2002. Expression and
function of histamine receptors in human monocyte-derived
dendritic cells. J. Allergy Clin. Immunol. 109:839.
H2R modulates monocyte-derived DC differentiation 115
by guest on November 24, 2015
29 Gutzmer, R., Diestel, C., Mommert, S. et al. 2005. Histamine
H4 receptor stimulation suppresses IL-12p.70 production and
mediates chemotaxis in human monocyte-derived dendritic cells.
J. Immunol. 174:5224.
30 Ohtani, T., Aiba, S., Mizuashi, M., Mollah, Z. U., Nakagawa, S. and
Tagami, H. 2003. H1 and H2 histamine receptors are absent on
Langerhans cells and present on dermal dendritic cells. J. Invest.
31 Dijkstra, D., Stark, H., Chazot, P. L. et al. 2008. Human
inflammatory dendritic epidermal cells express a functional
histamine H4 receptor. J. Invest. Dermatol. 128:1696.
32 Gschwandtner, M., Rossbach, K., Dijkstra, D. et al. 2010. Murine
and human Langerhans cells express a functional histamine H4
receptor: modulation of cell migration and function. Allergy
33 Mazzoni, A., Young, H. A., Spitzer, J. H., Visintin, A. and Segal, D.
M. 2001. Histamine regulates cytokine production in maturing
dendritic cells, resulting in altered T cell polarization. J. Clin.
34 Jawdat, D. M., Albert, E. J., Rowden, G., Haidl, I. D. and Marshall,
J. S. 2004. IgE-mediated mast cell activation induces Langerhans
cell migration in vivo. J. Immunol. 173:5275.
35 Dawicki, W., Jawdat, D. W., Xu, N. and Marshall, J. S. 2010. Mast
cells, histamine, and IL-6 regulate the selective influx of dendritic
cell subsets into an inflamed lymph node. J. Immunol. 184:2116.
36 Baumer, W., Wendorff, S., Gutzmer, R. et al. 2008. Histamine
H4 receptors modulate dendritic cell migration through skin–
immunomodulatory role of histamine. Allergy 63:1387.
37 Cowden, J. M., Zhang, M., Dunford, P. J. and Thurmond, R. L.
2010. The histamine H4 receptor mediates inflammation and
pruritus in Th2-dependent dermal inflammation. J. Invest. Derma-
38 Dijkstra, D., Leurs, R., Chazot, P. et al. 2007. Histamine down-
regulates monocyte CCL2 production through the histamine H4
receptor. J. Allergy Clin. Immunol. 120:300.
39 Dunford, P. J., O’Donnell, N., Riley, J. P., Williams, K. N., Karlsson,
L. and Thurmond, R. L. 2006. The histamine H4 receptor mediates
allergic airway inflammation by regulating the activation of CD4+ T
cells. J. Immunol. 176:7062.
40 de Saint-Vis, B., Fugier-Vivier, I., Massacrier, C. et al. 1998. The
cytokine profile expressed by human dendritic cells is dependent
on cell subtype and mode of activation. J. Immunol. 160:1666.
41 Tetlow, L. C. and Woolley, D. E. 2002. Histamine stimulates matrix
metalloproteinase-3 and -13 production by human articular
chondrocytes in vitro. Ann. Rheum. Dis. 61:737.
42 Del Prete, A., Locati, M., Otero, K. et al. 2006. Migration of
dendritic cells across blood and lymphatic endothelial barriers.
Thromb. Haemost. 95:22.
43 Randolph, G. J., Sanchez-Schmitz, G. and Angeli, V. 2005.
Factors and signals that govern the migration of dendritic cells via
lymphatics: recent advances. Springer Semin. Immunopathol.
44 Schiefner, A. and Wilson, I. A. 2009. Presentation of lipid antigens
by CD1 glycoproteins. Curr. Pharm. Des. 15:3311.
45 Katoh, N., Soga, F., Nara, T., Masuda, K. and Kishimoto, S. 2005.
Histamine induces the generation of monocyte-derived dendritic
cells that express CD14 but not CD1a. J. Invest. Dermatol.
46 Caron, G., Delneste, Y., Roelandts, E. et al. 2001. Histamine
polarizes human dendritic cells into Th2 cell-promoting effector
dendritic cells. J. Immunol. 167:3682.
47 Chang, C. C., Wright, A. and Punnonen, J. 2000. Monocyte-
derived CD1a+ and CD1a- dendritic cell subsets differ in their
cytokine production profiles, susceptibilities to transfection, and
capacities to direct Th cell differentiation. J. Immunol. 165:3584.
48 Simon, T., Laszlo, V., Lang, O., Buzas, E. and Falus, A. 2011.
Histamine regulates relevant murine dendritic cell functions via H4
receptor. Front. Biosci. (Elite Ed.) 3:1414.
49 Osman, M., Tortorella, M., Londei, M. and Quaratino, S. 2002.
Expression of matrix metalloproteinases and tissue inhibitors of
metalloproteinases define the migratory characteristics of human
monocyte-derived dendritic cells. Immunology 105:73.
50 Bracke, K., Cataldo, D., Maes, T. et al. 2005. Matrix metalloproteinase-
12 and cathepsin D expression in pulmonary macrophages and
dendritic cells of cigarette smoke-exposed mice. Int. Arch. Allergy
51 Schakel, K., Mayer, E., Federle, C., Schmitz, M., Riethmuller, G.
and Rieber, E. P. 1998. A novel dendritic cell population in human
blood: one-step immunomagnetic isolation by a specific mAb (M-
DC8) and in vitro priming of cytotoxic T lymphocytes. Eur. J.
52 Li, K., Fazekasova, H., Wang, N. et al. 2011. Expression of
complement components, receptors and regulators by human
dendritic cells. Mol. Immunol. 48:1121.
53 Weaver, D. J. Jr, Reis, E. S., Pandey, M. K. et al. 2010. C5a
receptor-deficient dendritic cells promote induction of Treg and
Th17 cells. Eur. J. Immunol. 40:710.
54 Sallusto, F., Schaerli, P., Loetscher, P. et al. 1998. Rapid and
coordinated switch in chemokine receptor expression during
dendritic cell maturation. Eur. J. Immunol. 28:2760.
55 Kabashima, K., Shiraishi, N., Sugita, K. et al. 2007. CXCL12-
CXCR4 engagement is required for migration of cutaneous
dendritic cells. Am. J. Pathol. 171:1249.
56 Chong, S. Z., Wong, K. L., Lin, G. et al. 2011. Human CD8 Tcells
drive Th1 responses through the differentiation of TNF/iNOS-
producing dendritic cells. Eur. J. Immunol. 41:1639.
57 Rogers, N. M., Matthews, T. J., Kausman, J. Y., Kitching, A. R. and
Coates, P. T. 2009. Review article: kidney dendritic cells: their role
in homeostasis, inflammation and transplantation. Nephrology
116H2R modulates monocyte-derived DC differentiation
by guest on November 24, 2015