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*
Division of Clinical Immunology and Allergy and
Section of Pharmacology, Department of Neuroscience, University of Naples Federico II, Naples, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-glucuronidase
(EC50 = 8.2 ± 3.5 x 10-9 M)
and IL-6 (EC50 = 9.3 ± 2.9 x
10-8 M) from human lung macrophages. Enhancement of
-glucuronidase release induced by histamine was evident after 30 min
and peaked at 90 min, whereas that of IL-6 required 2–6 h of
incubation. These effects were reproduced by the H1 agonist
(6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane
carboxamide but not by the H2 agonist dimaprit.
Furthermore, histamine induced a concentration-dependent increase of
intracellular Ca2+ concentrations
([Ca2+]i) that followed three types of
response, one characterized by a rapid increase, a second in which
[Ca2+]i displays a slow but progressive
increase, and a third characterized by an oscillatory pattern.
Histamine-induced -glucuronidase and IL-6 release and
[Ca2+]i elevation were inhibited by the
selective H1 antagonist fexofenadine
(10-7–10-4 M), but not by the H2
antagonist ranitidine. Inhibition of histamine-induced
-glucuronidase and IL-6 release by fexofenadine was concentration
dependent and displayed the characteristics of a competitive antagonism
(Kd = 89 nM). These data demonstrate
that histamine induces exocytosis and IL-6 production from human
macrophages by activating H1 receptor and by increasing
[Ca2+]i and they suggest that histamine may
play a relevant role in the long-term sustainment of allergic
inflammation in the airways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increasing evidence suggests that histamine not only is massively released during acute allergic reactions, but can be secreted in low concentrations in the airways of patients with bronchial asthma. In fact, detectable levels of histamine have been found in the bronchoalveolar lavage (BAL)3 of patients with bronchial asthma during asymptomatic periods (14, 15, 16), and these levels have been found to correlate with asthma severity and bronchial hyperreactivity (17). In addition, basophils and BAL mast cells from patients with asthma have an increased capacity to release histamine in vitro as compared with those from healthy donors (14). Finally, an increased number of degranulated mast cells and basophils also has been documented in the airways of asthmatics when biopsies were performed at a time distant from the acute attack (18, 19). These observations are compatible with the hypothesis that histamine is chronically released in the airways of asthmatic patients and may have a role in persistent airway inflammation and tissue remodeling typical of bronchial asthma (20).
The macrophage is the predominant cell in the lung parenchyma and in the BAL of both healthy individuals and asthmatic patients (21). This cell plays a major role in the defense against infections and in the local modulation of immune and inflammatory responses (22). Lung macrophages produce a wide spectrum of mediators including lytic enzymes, lipid mediators, reactive oxygen radicals, cytokines, and chemokines (23). These cells can be activated by a variety of stimuli acting on specific membrane receptors including the three histamine receptors (H1, H2 and H3; Refs. 24 and 25). Activation of histamine receptors modulates several macrophage functions such as the expression of adhesion molecules (26) and exocytosis (24). Macrophages are often found in close proximity to mast cells in the airways of asthmatic patients (18, 19). The anatomical association between these two cells suggests that lung macrophages may be exposed to histamine released locally from immunologically activated mast cells.
In this study, we have examined the effect of low concentrations of
histamine on human lung macrophages in vitro. Our results indicate that
histamine induces the release of -glucuronidase, a marker of
exocytosis, and the production of IL-6 by activating the
H1 receptor and by increasing the intracellular
Ca2+ concentrations
([Ca2+]i). These effects
are reproduced by selective H1 receptor
activation and are inhibited in a competitive fashion by fexofenadine,
a second generation H1 receptor antagonist.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histamine dihydrochloride, ranitidine hydrochloride, Percoll,
L-glutamine, antibiotic-antimycotic solution (10,000 IU/ml
penicillin, 10 mg/ml streptomycin, and 25 µg/ml amphotericin B),
group IA secretory phospholipase A2
(PLA2) (from Naja mossambica
mossambica), LPS (from Escherichia coli serotype
026:B6), fatty acid-free human serum albumin, Triton X-100,
1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(21-amino-51-methylphenoxy)-ethane-N,N,N1,N1-tetraacetic
acid pentaacetoxymethyl ester (fura-2 AM), and
phenolphthalein glucuronide were purchased from Sigma (St. Louis, MO).
(6-[2-(4-Imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane
carboxamide (HTMT dimaleate) and dimaprit dihydrochloride were
purchased from Tocris Cookson Ltd. (Bristol, U.K.). Platelet-activating
factor was purchased from Biomol (Plymouth, PA). A23187 was purchased
from Calbiochem (La Jolla, CA). RPMI 1640 and FCS were purchased from
ICN Pharmaceuticals (Costa Mesa, CA). Fexofenadine was a gift from Dr.
Patrizia Pugnetti (Aventis, Milan, Italy). A 100-bp DNA ladder was
purchased from MBI Fermentas (Vilnius, Lituania). IL-6 and -actin
primers were designed by Dr. David Essayan (Johns Hopkins University,
Baltimore, MD) and were produced and purified by the Johns Hopkins DNA
Core Facility. All other reagents were obtained from Carlo Erba (Milan,
Italy).
PIPES buffer was composed of 25 mM PIPES (Sigma), 110 mM NaCl, and 5 mM KCl, pH 7.4. Glycine buffer was composed of 400 mM glycine and 400 mM NaCl, pH 10.3. Krebs-Ringer saline solution for Ca2+ experiments was made up of 160 mM NaCl, 5.5 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4.
Isolation and purification of human lung macrophages
Macrophages were obtained from the lung parenchyma of patients
undergoing thoracic surgery as reported previously (27).
Macroscopically normal tissue was minced finely with scissors and
washed extensively with PIPES buffer over Nytex cloth (120-µm pore
size; Tetko, Elmsford, NY). The macrophage suspension was enriched
(75–85%) by flotation over Percoll density gradients, as described
previously (27), and the cells were resuspended
(106 cells/ml) in RPMI 1640 containing 5% FCS, 2
mM L-glutamine, and 1% antibiotic-antimycotic solution.
The cells then were incubated in 24-well plates (Falcon; Becton
Dickinson, Franklin Lakes, NJ) at 37°C in a humidified atmosphere of
5% CO2 and 95% air. After 12 h, the medium
was removed and the plates were gently washed three times with RPMI
1640. Adherent cells were found to be >98% macrophages as assessed by
-naphthyl acetate esterase staining (27).
Cell incubations
Macrophages adherent to 24-well plates were incubated (37°C,
30 min to 18 h) in RPMI 1640 containing various concentrations of
freshly prepared histamine, HTMT (an H1 agonist),
or dimaprit (H2 agonist). In some experiments,
the cells were preincubated (37°C, 15 min, unless otherwise
specified) with various concentrations of fexofenadine or ranitidine
before the addition of histamine. At the end of the experiment, the
supernatant was removed, centrifuged twice (1000 x g,
4°C, 5 min), and stored up to 72 h at -80°C for subsequent
determination of -glucuronidase and IL-6. At the end of each
experiment, an aliquot of cells was stained with trypan blue to
determine cell viability. The cells remaining in the plates were lysed
with 0.1% Triton X-100 for the determination of total cellular content
of proteins and -glucuronidase.
-Glucuronidase assay
-Glucuronidase release in cell-free supernatants or in cell
lysates was measured by a colorimetric assay (28).
Briefly, 200 µl of supernatants or 100 µl of cell lysates was
incubated (37°C, 18 h) with 400 µl of 0.1 M acetate buffer, pH
4.5, containing 1 µmol of phenolphthalein glucuronide. At the end of
the incubation, 2 ml of glycine buffer was added to each tube and the
mixture was transferred into 3-ml cuvette for OD reading at 540 nm.
-Glucuronidase release was expressed as the percentage of the total
cellular content, determined in cells lysed with 0.1% Triton X-100.
All experiments were conducted in duplicate determinations.
IL-6 ELISA
IL-6 in the culture supernatant of macrophages was measured in duplicate determinations by a commercially available ELISA (Euro Clone, Torquay, U.K.) according to the manufacturer’s instructions. The linearity range of the assays was between 5 and 150 pg/ml. Because the number of adherent macrophages can vary in each well and in different experiments, the results were normalized for the total protein content in each well, determined in the cell lysates (0.1% Triton X-100) by the method of Lowry et al. (29).
IL-6 gene expression
Macrophages (5 x 106/2 ml) were
incubated (37°C, 1 h) in RPMI 1640 containing 5% FCS in
six-well plates. The cells then were washed and incubated (37°C, 1–9
h) in FCS-free medium alone or with histamine
(10-6 M). At the end of the incubation, RNA was
isolated with the TRIzol reagent (Life Technologies, Grand Island, NY),
according to the manufacturer’s instructions.
Diethylpyrocarbonate-treated water without SDS was used for the final
resuspension step; RNA was stored at -80°C. Reverse transcription
was performed with 5 mM MgCl2,
oligo(dT)16 primer, and murine leukemia virus
reverse transcriptase according to the manufacturer’s instructions
(Perkin-Elmer, Norwalk, CT) on a thermocycler (GeneAmp PCR System 2400;
Perkin-Elmer). PCR was performed by using Taq
polymerase (1–2.5 U/reaction) at the annealing temperature of 60°C
with target-specific primers for IL-6
(5'-ATGAACTCCTTCTCCACAAGCGC-3' and 3'-GAAGAGCCCTCAGGCTGGACTG-5' at
0.2–1 µM/primer) at subsaturating cycle number (30 cycles).
Normalization of RNA was achieved by RT-PCR for the constitutive marker
gene -actin at subsaturating cycle number. Strict RNase-free
conditions were maintained throughout the procedure (30).
All PCR products were visualized by ethidium bromide-stained gel
electrophoresis and photographed. Analysis of relative band intensity
was performed using a digital scanner (Celbio, Milan, Italy).
[Ca2+]i measurements and analysis of [Ca2+]i oscillations
[Ca2+]i was measured by a microfluorometric technique, as reported previously (31). Briefly, the cells grown on glass coverslips were loaded with 5 µM fura-2 AM in Krebs-Ringer saline solution for 1 h at 22°C. At the end of fura-2 AM loading, the coverslip was introduced into a microscope chamber (Medical System, Greenvale, NY) on an inverted Nikon Diaphot fluorescence microscope (Nikon, Melville, NY). The cells were kept in Krebs-Ringer saline solution throughout the experiment. All the drugs tested were introduced into the microscope chamber by fast injection. A 100-watt xenon lamp (Osram, Berlin, Germany) with a computer-operated filter wheel bearing two different interference filters (340 and 380 nm) illuminated the microscopic field with UV light, alternating the wavelength at an interval of 500 ms. The interval between each pair of illuminations was 2 s, and the interval between filter movements was 1 s. Consequently, [Ca2+]i was measured every 3 s. Emitted light was passed through a 400-nm dichroic mirror, filtered at 510 nm, and collected by a charge-coupled device camera (Photonic Science, Robertsbridge, East Sussex, U.K.) connected to a light amplifier (Applied Imaging, Dukesway Gateshead, U.K.). Images were digitized and analyzed with a Magiscan image processor (Applied Imaging). By using a calibration curve, the Tardis software (Applied Imaging) calculated the [Ca2+]i corresponding to each pair of images from the ratio between the intensity of the light emitted when the cells were illuminated at both 340 and 380 nm.
[Ca2+]i oscillations were defined as an increase of [Ca2+]i above the mean of the basal value ± 2 SD. The frequency of [Ca2+]i oscillations was calculated as the number of peaks occurring per min, and the amplitude was determined as the difference between the maximal [Ca2+]i value of the peak and the minimal [Ca2+]i value just before the occurrence of the peak.
Statistical analysis
The data are expressed as the mean ± SE of the indicated number of experiments, and p values were determined with t test for unpaired samples with Bonferroni’s correction (32). The threshold for statistical significance was set at p < 0.05. The data subjected to linear regression analysis were calculated by the least squares method (y = a + bx) in which a was the y-axis intercept and b the slope of the line. For each pharmacological treatment in the experiments on [Ca2+]i, at least five cells in at least three experimental sessions were evaluated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glucuronidase release from human lung
macrophages
We first examined whether histamine induced the release of
-glucuronidase, a marker of exocytosis (33), from human
lung macrophages. Fig. 1
shows the
results of six experiments indicating that histamine induces a
concentration-dependent release of -glucuronidase from macrophages.
After 2 h of incubation, a significant effect of histamine was
evident at a concentration of 10-9 M, whereas
the maximum release of -glucuronidase occurred at
10-7 M, with an EC50 of
8.2 ± 3.5 x 10-9 M. The highest
concentration of histamine (10-7 M) induced a
release that was 2.4-fold higher than that from unstimulated cells
(7.2 ± 0.5 vs 3.0 ± 0.3% of total cellular content;
p < 0.01). Concentrations of histamine higher than
10-7 M did not further enhance -glucuronidase
release. Viability of macrophages was routinely assessed at the end of
each experiment by trypan blue exclusion and was found to be always
>95%.
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shows the kinetics of histamine
(10-7 M)-induced -glucuronidase release from
macrophages. The release was relatively rapid, starting after 30 min
and reaching a plateau 2 h after the addition of the stimulus.
These data indicate that submicromolar concentrations of histamine
rapidly enhance -glucuronidase release from macrophages.
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Macrophages are an important source of cytokines regulating
inflammatory and immune responses in the lung. Although histamine has
been reported either to induce (25, 34) or inhibit
(35, 36) cytokine synthesis in human monocytes and
macrophages, there are no data on the effect of this mediator on IL-6,
a major cytokine produced by human macrophages (37). In
these experiments, macrophages were incubated with increasing
concentrations of histamine ranging from 10-9 to
10-6 M for 6 h at 37°C. Histamine
increased the basal secretion of IL-6 from macrophages in a
concentration-dependent fashion (Fig. 3)
with a maximum enhancement of 2-fold the basal release (1.6 ± 0.3
vs 0.8 ± 0.1 ng of IL-6/mg of protein) at
10-6 M. Histamine also appeared to be less
effective in inducing IL-6 release than it was on -glucuronidase
because a significant effect on IL-6 release was achieved only at a
concentration of 10-7 M and the
EC50 was 9.3 ± 2.9 x
10-8 M.
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shows the kinetics of IL-6 release
from three preparations of human lung macrophages challenged with
10-6 M of histamine. Maximum enhancement of IL-6
release was evident between 2 and 6 h of incubation and the
average half-time for IL-6 enhancement was 3.4 ± 0.8
h.
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). The results indicate
that histamine is the less potent stimulus; it induced a production of
IL-6 that was approximately one-half of that induced by other
stimuli.
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-glucuronidase and IL-6 release
from human lung macrophages
To determine the type of receptor activated by histamine on
macrophages, the cells were incubated (37°C, 2 h for
-glucuronidase and 6 h for IL-6) with either HTMT, a selective
H1 agonist (38), or dimaprit, a
selective H2 agonist (39). Table I shows that HTMT but not dimaprit
induces the release of both -glucuronidase and IL-6 from
macrophages. HTMT had an effect comparable to that of histamine as it
increased 
2-fold the basal release of both -glucuronidase and
IL-6. These data indicated that activation of the
H1 receptor was involved in histamine-induced
exocytosis and IL-6 release.
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-glucuronidase was
determined. Fig. 6A shows that
increasing concentration of fexofenadine
(10-7–10-4 M) induced a
parallel rightward shift of the histamine concentration-response curve.
In addition, the inhibitory effect of fexofenadine was progressively
reduced by increasing the concentrations of the agonist (histamine).
This observation suggested that fexofenadine acted as a competitive
antagonist at the receptor level. In contrast, ranitidine
(10-4 M) had no effect on histamine-induced
-glucuronidase release.
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A were used
for Schild plot analysis. In case of a competitive antagonism, Schild
plot analysis provides a straight line with an intercept on the
abscissa corresponding to log Kd. Fig. 6B shows the least squares fit to the experimental points
from four separate experiments. The mean ± SE value for
Kd of fexofenadine was 89.2 ±
22.6 nM, a value that is close to the
Ki reported for fexofenadine in
binding the H1 receptor in rat brain tissue (160
nM; Ref. 41).
Additional experiments were performed to determine whether the
H1 antagonist fexofenadine also inhibited
histamine-induced IL-6 release. Fig. 7
shows that preincubation of macrophages with fexofenadine for 15 min
inhibits in a concentration-dependent fashion the release of IL-6
induced by 10-7 M histamine. The inhibitory
effect of fexofenadine was significant at 10-6
M, and it reached a maximum inhibition of 85 ± 15% of histamine
response at a concentration of 10-4 M. Together
these data indicate that both -glucuronidase and IL-6 release are
mediated by the activation of the H1 receptor on
macrophages.
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Activation of the H1 receptor is associated with phospholipase C activation and inositol 1,4,5-triphosphate generation leading to a raise in [Ca2+]i (42). The aforementioned results indicated that human macrophages express a functionally active H1 receptor. Therefore, in the next group of experiments, we monitored with the help of a microfluorometric technique at a single-cell level the histamine-induced changes in [Ca2+]i.
When human lung macrophages were exposed to 10-7
M histamine, an elevation of
[Ca2+]i occurred in
45% of the cells examined. Moreover, responsive macrophages
displayed three different patterns of
[Ca2+]i elevation (Fig. 8A). Sixty-five percent of the
cells exhibited a rapid and long-lasting elevation of baseline
[Ca2+]i (Fig. 7A, upper tracing). In a second subgroup of cells
(23%), histamine response was characterized by a slower but
progressive increase of
[Ca2+]i (Fig. 8A, middle tracing). Interestingly, in the
remaining 11% of lung macrophages an oscillatory pattern was elicited
by histamine (Fig. 8A, lower tracing). In these
cells, histamine-induced
[Ca2+]i oscillations were
characterized by a frequency of 1–3/min and an amplitude ranging from
40 to 60 nM. Histamine-induced
[Ca2+]i elevation in
responsive macrophages was concentration dependent and reached a
maximum increase of 80% as compared with baseline at
10-6 M (Fig. 8B).
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A). The
concentration-response curve of the inhibitory effect of fexofenadine
on histamine-induced
[Ca2+]i changes is shown
in Fig. 9B.
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The data reported above indicated that histamine induced IL-6
release and increased
[Ca2+]i in human lung
macrophages. To understand whether the increase in
[Ca2+]i was required for
IL-6 release from macrophages, the cells were stimulated with histamine
either in the absence or in the presence of the
Ca2+ chelating agent EDTA (10 mM). Table II shows that incubation of macrophages
with EDTA completely inhibits IL-6 release in response to histamine but
not the spontaneous release. In separate experiments we explored
whether the Ca2+ ionophore A23187 induces IL-6
release from macrophages. In three experiments, A23187
(10-6 M) significantly increased IL-6 release
from human lung macrophages (2.51 ± 0.26 vs 0.92 ± 0.19
ng/mg of protein; p < 0.05). These data are compatible
with the hypothesis that the increase in
[Ca2+]i is necessary for
histamine-induced IL-6 release.
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It has been reported that Ca2+ signals
differentially activate nuclear transcription factors depending on
their amplitude, duration, and oscillatory pattern (43, 44). To test the hypothesis that histamine may activate gene
expression for IL-6, we evaluated the expression of IL-6 mRNA in
macrophages incubated with medium alone (control) or with histamine
(10-6 M) for 1–9 h. Fig. 10A depicts specific RT-PCR
amplification products from one representative experiment of three.
Adequate normalization of RNA for each sample was confirmed by the
equality of RT-PCR amplification products for the constitutive marker
gene of -actin (first row). Histamine increased
IL-6 mRNA expression after 3 and 6 h of incubation. The enhancing
effect of histamine was no longer evident after 9 h of incubation.
Fig. 10B shows the densitometric analysis of the IL-6 bands,
expressed as the ratio of the signal in histamine-treated and
-untreated cells. These data indicate that histamine enhances IL-6
production in macrophages by increasing its specific mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both experimental and clinical evidence suggests that low levels of
histamine are chronically released in the airways of asthmatic patients
even in the absence of clinical symptoms (14, 15, 16, 17). The
level of histamine detected in the BAL fluid of asthmatics is 1–2
ng/ml (15, 16). Considering the dilution of the BAL and
the intimate contact between mast cells and macrophages in the airways,
the concentrations of histamine surrounding lung macrophages may reach
10-7–10-6 M. Our results
demonstrate that these concentrations of histamine are effective to
increase exocytosis and IL-6 release from macrophages.
Our data provide the first evidence that physiologically relevant concentrations of histamine induce the release of IL-6 from human macrophages isolated from the lung parenchyma. Histamine increases IL-6 production presumably by increasing its mRNA expression. However, our data do not exclude the possibility that histamine increases the stability of IL-6 mRNA. IL-6 is a multifunctional cytokine involved in inflammatory and immune responses (37). Previous studies have shown that the levels of IL-6 are increased in plasma and BAL (45, 46, 47) and that this cytokine is overexpressed in the bronchial mucosa of patients with bronchial asthma (48). Although its role has not been completely elucidated, IL-6 may participate to allergic inflammation in a number of ways. IL-6 is a stem cell factor and a B cell proliferating and activating factor (37). For example, IL-6 is involved in mast cell proliferation and differentiation (49, 50), it may favor Th2 responses and IgE production (51, 52) and stimulates airway epithelial cells to release IL-8 (53). These observations support the hypothesis that IL-6 may have an important role in modulating allergic inflammation in asthma. Our results are in agreement with previous data showing that histamine induces IL-6 production in bronchial epithelial cells (54), endothelial cells (55), and B cells (56). Therefore, histamine appears to activate a common pathway leading to IL-6 expression in all the major sources of this cytokine in the human lung.
The conclusion that histamine induces exocytosis and IL-6 production
through the activation of H1 receptors on
macrophages is supported by two lines of evidence: 1) these events are
induced by HTMT, a selective H1 agonist, but not
by the H2 agonist dimaprit, and 2) they are
inhibited by the H1 antagonist fexofenadine but
not by the H2 antagonist ranitidine. Human
macrophages express all types of histamine receptors,
H1, H2, and
H3 (24, 25). Several studies have
started to highlight the complexity of histamine’s effects on cytokine
network in human cells depending on the type of receptor activated. For
example, activation of IL-6 production generally occurs via
H1 receptors (54, 55, 56), whereas
inhibition of IL-1, TNF-, and IL-12 production by LPS-stimulated
human monocytes is mediated by H2 receptors
(35, 36, 57). Furthermore, activation of
H2 and H3 receptors
stimulates IL-10 release from human monocytes and macrophages
(11, 25). These findings suggest that the local release of
histamine in inflamed tissues may play a role in the modulation of the
cytokine network more complex than originally thought. Even more
interesting is the hypothesis that histamine may differentially
modulate cytokine synthesis depending on the type of receptor
predominantly expressed on a given cell. Studies are currently ongoing
to define whether macrophages isolated from patients with bronchial
asthma express a pattern of histamine receptors different from those of
nonasthmatic individuals.
Stimulation of macrophages with histamine results in the increase in [Ca2+]i, and this effect is inhibited by the H1 competitive antagonist fexofenadine. These results are in line with the general observation that H1 receptor activation is associated with intracellular Ca2+ influx (39) and they indicate that this signaling pathway is also active in human macrophages. Moreover, our results suggest that the increase in [Ca2+]i induced by histamine is required for the activation of IL-6 production in these cells.
A number of studies support the hypothesis that subsets of macrophages
with different morphological, biochemical, and functional properties
exist in the human lung (58). Whether these differences
are related to a different state of maturation or activation of
macrophages is presently unclear. We add a novel observation to support
the hypothesis of lung macrophage heterogeneity by showing that the
same concentration of histamine may induce three distinct profiles of
Ca2+ response in these cells: a slow increase, a
rapid increase, and a series of phasic oscillations. These different
profiles of Ca2+ response may reflect either the
activation of diverse macrophage populations or qualitative differences
in the expression of histamine receptors on macrophages. In any event,
lung macrophages display a heterogenous Ca2+
response to histamine. This finding acquires further relevance in light
of the recent demonstration that different profiles of
Ca2+ signaling may selectively activate nuclear
transcription factors such as NF-
B, c-Jun N-terminal kinase, and
NF-AT (43, 44). Therefore, the induction of different
Ca2+ responses may be a mechanism by which
histamine modulates the expression of various cytokines in the human
macrophages.
The second generation H1 antagonist fexofenadine inhibits histamine-induced exocytosis, IL-6 production, and Ca2+ signaling. The observation that fexofenadine is a competitive antagonist at the histamine H1 receptor level on human lung macrophage with a Kd comparable to that obtained in other tissues is of interest for a number of reasons. First, it indicates that the H1 receptor on macrophages displays pharmacological characteristics similar to the H1 receptor expressed in other tissues. Second, the observation that the Kd of fexofenadine is close to the Ki indicates that the inhibitory effect of this drug is truly a pharmacological event at the receptor level and it does not represents a nonspecific interaction of fexofenadine with the cell membrane. Finally, the observation that fexofenadine not only antagonizes the exocytotic effect of histamine on macrophages, but also the synthesis of IL-6 is of potential clinical importance. In fact, it suggests that administration of this drug in patients with allergic disorders may prevent not only the acute symptoms, but that it might interfere with some of the mechanisms involved in chronic inflammation and in tissue damage associated with the activation of macrophages.
Taken together, our data demonstrate that histamine activates human
lung macrophages via H1 receptor. In these cells,
histamine enhances the release of a preformed mediator
(-glucuronidase) as well as the expression and release of IL-6.
These effect of histamine are associated with a raise in cytosolic
Ca2+ concentrations. These novel actions of
histamine on a cell that plays a central role in inflammatory lung
diseases provide an additional mechanism by which histamine contributes
to maintain chronic inflammation in bronchial asthma. The ability of
fexofenadine, a selective H1 blocker, to inhibit
histamine-induced activation of human macrophages opens new
perspectives on the long-term use of H1 receptor
antagonists in the treatment of allergic and inflammatory lung
diseases.
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Footnotes |
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2 Address correspondence and reprint request to Dr. Massimo Triggiani, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy.
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage; fura-2 AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(21-amino-51-methylphenoxy)-ethane-N,N,N1,N1-tetraacetic acid pentaacetoxymethyl ester; HTMT, (6-[2-(4-im-idazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide; [Ca2+]i, intracellular Ca2+ concentration; PLA2, phospholipase A2.
Received for publication August 7, 2000. Accepted for publication January 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glucuronidase release from human lung macrophages. Am. J. Respir. Cell. Mol. Biol. 3:603.
-glucuronidase release and IL-6 production from human lung macrophages. J. Immunol. 164:4908. via histamine H2 receptors. J. Exp. Med. 174:281.- and interleukin-6-triggered mast cell development from mouse spleen cells. Blood 89:526., IL-6, and Il-12 production. Eur. Respir. J. 114:106.
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 U. W Khan and U. Rai Differential effects of histamine on Leydig cell and testicular macrophage activities in wall lizards: precise role of H1/H2 receptor subtypes J. Endocrinol., August 1, 2007; 194(2): 441 - 448. [Abstract] [Full Text] [PDF]
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 A. Wimmer, S. K. Khaldoyanidi, M. Judex, N. Serobyan, R. G. DiScipio, and I. U. Schraufstatter CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes Blood, December 1, 2006; 108(12): 3722 - 3729. [Abstract] [Full Text] [PDF]
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) or with histamine (10-7 M; •). At each
time point, supernatants were collected and centrifuged (1000 x
g, 4°C, 5 min). 
) or with increasing concentration of fexofenadine
(10-7 M, 
; 10-5
M, 
; 10-4 M, •) or ranitidine (10-4 M,
) and were then incubated (2 h, 37°C) with histamine
(10-7 M). At the end of the incubation, the supernatant
was collected and centrifuged (1000 x g, 4°C, 5
min). 
,
p < 0.05 vs histamine alone.
