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Originally published online as doi:10.2353/ajpath.2009.080871 on March 5, 2009

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(American Journal of Pathology. 2009;174:1426-1434.)
© 2009 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.080871

Histamine Promotes Osteoclastogenesis through the Differential Expression of Histamine Receptors on Osteoclasts and Osteoblasts

Martin Biosse-Duplan*, Brigitte Baroukh*, Michel Dy{dagger}, Marie-Christine de Vernejoul{ddagger} and Jean-Louis Saffar*

From the EA 2496,* and the UMR 8147,{dagger} CNRS, Université Paris Descartes, Paris; and INSERM U606,{ddagger} Université Paris Diderot, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to the numerous roles of histamine in both the immune and nervous systems, previous studies have suggested that this bioamine might also be involved in bone metabolism. Following our observations of impaired bone resorption in ovariectomized rats after histamine receptor antagonist treatment, we focused in this study on osteoclasts and osteoclast precursors. We looked for a direct action of histamine on these cells using both in vivo and in vitro approaches. In vivo, we triggered a remodeling sequence in rat mandibular bone and treated the animals with either histamine or histamine receptor antagonists. Histamine was shown to increase the number of osteoclasts and osteoclast precursors whereas antagonists of histamine receptor-1 and -2 decreased both osteoclast recruitment and resorption. In vitro, spleen cells from histamine-deficient mice were treated with receptor activator for nuclear factor kappa B ligand and macrophage colony stimulating factor, giving rise to both reduced numbers of osteoclasts and decreased resorption on dentin slices. Histamine enhanced resorption in these cultures in a dose-dependent manner. In addition, we identified osteoclast precursors as a source of histamine. In contrast, histamine increased the receptor activator for nuclear factor kappa B ligand/osteoprotegerin ratio in primary osteoblasts that did not secrete histamine. We observed a differential expression of histamine receptor-1 and -2 mRNAs in both primary osteoclasts and osteoblasts, confirming their functional roles with selective antagonists. Thus, histamine acts directly on osteoclasts, osteoclast precursors, and osteoblasts, promoting osteoclastogenesis through autocrine/paracrine mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone remodeling is the coordinated process that continuously renews a small amount of mineralized tissue throughout the skeleton to maintain an optimum bone structure adapted to mechanical and metabolic demands. It involves a coupling of resorption of mineralized bone by osteoclasts and the deposition by osteoblasts of bone matrix that subsequently becomes mineralized. Excessive resorption leads to bone loss, as it occurs in osteoporosis, Paget’s bone disease, tumor osteolysis, some arthritis, and periodontitis. Osteoclasts are derived from hematopoietic stem cells of the monocyte/macrophage lineage1 while osteoblasts originate from pluripotent stromal stem cells.2 Molecular communication between osteoblasts and osteoclasts and between bone cells and other bone marrow cells is a fundamental mechanism that regulates the commitment, proliferation, and differentiation of bone cell precursors. The main osteoclastogenic signal is the receptor activator for NF-{kappa}B-ligand (RANKL), a cytokine that is expressed by osteoblasts. Its action on the RANK receptor is modulated by osteoprotegerin (OPG), a decoy receptor, which is also derived from osteoblasts.3 Conversely, activated osteoclasts may in turn influence osteoblasts and bone formation.4 In addition to the cross talk between bone cells, bone marrow cells are responsible for the production of paracrine and autocrine bone regulatory factors required for normal osteoclastogenesis.5

Histamine is a bioamine whose role has mainly been investigated as an important mediator of inflammatory and allergic responses, and as a neurotransmitter in the brain. Histamine is stored by two cell types, namely the mast cells and the basophiles; other immune cell lineages also synthesize and immediately release histamine in response to various stimuli.6 Histamine is synthesized from histidine by an enzyme (L-histidine decarboxylase, HDC) and exerts its effects by binding on four specific G-protein-coupled receptors, histamine receptors -1 to -4 (H1R to H4R). These receptors are expressed either ubiquitously (H1R and H2R) or restricted to specialized cell populations (H3R in the brain, H4R on hematopoietic cells).

Several observations have suggested that histamine may also be involved in bone metabolism. Mast cells are present close to the periosteum and the bone surface,7 and systemic mastocytosis, a disease characterized by infiltration of bone marrow and other tissues by mast cells, can lead to severe osteoporosis.8,9 Hematopoietic populations in the bone marrow also produce histamine.10 Inhibition of H2R by an antagonist partially prevents the trabecular bone loss following ovariectomy.11 The H2R antagonist cimetidine also inhibits the articular osteopenia in rats with adjuvant-induced arthritis.12 HDC deficiency in mice results in increased bone formation and reduced bone resorption, and protects against ovariectomy-induced bone loss.13 It was shown in vitro, in bone marrow cultures or co-cultures, that histamine can increase osteoclast differentiation and that H1R and H2R antagonists partially inhibit histamine increase.12,14,15 Also, it was demonstrated that osteoblasts express H1R and H2R and that histamine increase RANKL expression in these cells.14,15

In this study, we focused on the osteoclast and the osteoclast precursors, and looked for a direct action of histamine on these cells, using in vivo and in vitro approaches. We assessed the effects of histamine deficiency and increase, and specific inhibition of histamine receptors on osteoclast differentiation and activity, and determined the histamine receptors implicated. We also investigated the potential cellular sources of histamine in the bone environment. Moreover, as osteoblasts regulate osteoclastogenesis through the production of RANKL and OPG, we analyzed the effect of histamine on the RANKL/OPG ratio. We show in this study, for the first time, that histamine has a direct action on osteoclast and osteoclast precursors and that osteoclastogenesis is regulated by histamine trough autocrine/paracrine mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and Reagents

Alpha-minimal essential medium ({alpha}-MEM) supplemented with L-glutamine (Invitrogen, Cergy-Pontoise, France), penicillin-streptomycin suspension (Invitrogen), and 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT) was used. Histamine, {alpha}-fluoromethylhistidine, compound 48/80 (c48/80), mepyramine, pyrilamine, famotidine, cimetidine, ciproxifan, JNJ 7777120, ascorbic acid, and 1,25-(OH)2VitD3 were purchased from Sigma-Aldrich Corp (Lyon, France). Human sRANKL and human recombinant macrophage colony stimulating factor (M-CSF) were purchased from Preprotech (Neuilly-Sur-Seine, France).

In Vivo Experimental Design

We used a synchronized model in which localized bone resorption is induced in rats along the periosteal surface of the buccal lower mandibular cortex, after the extractions of the antagonist upper maxillary molars.16 The lack of antagonist teeth leads to the egress of the lower right mandibular molars and the nontraumatic induction of a synchronous resorption sequence along the periosteum.17 The timing of the resorption wave has been extensively studied; 9 hours after induction (extractions) mast cells located close to the bone surface are activated and inflammatory cells, in particular monocytes expressing the ED1 marker, are recruited from the circulation. The recruitment of the monocytes reaches a maximum level 12 hours after induction and is complete 24 hours after induction; osteoclastic resorption follows the recruitment of the monocyte and peaks 4 days after induction.7,18

Rats were locally treated by injecting histamine (4 µl of a solution at 10 µg/ml), mast cell degranulating agent c48/80 (4 µl of a solution at 100 µg/ml) or saline (vehicle, VEH) close to the site of resorption19 8 hours after activation. Other rats were treated systemically with intramuscular injection of saline H1R antagonist mepyramine solution (1.5 mg/kg/day), saline H2R antagonist famotidine solution (10 mg/kg/day) or VEH, either beginning immediately after extractions (early treatment) or 24 hours later, ie, after inflammatory cell recruitment (delayed treatment). In one additional group (n = 6), histamine was locally injected without previous extraction. Food (M25 Extralabo; U.A.R., Villemoisson, France) and water were given ad libitum. Before extraction and sacrificing, rats were anesthetized with 8% chloral hydrate solution (Prolabo, Fontenay, France) injected intraperitoneally. The rats were used in compliance with European Union recommendations on laboratory animal care.

Tissue Processing, Immunohistochemistry, and Enzymohistochemistry

The right hemi mandibles were immediately removed, fixed for 24 hours in cold (4°C) 40% ethanol, gradually dehydrated, embedded without prior demineralization in methyl methacrylate (Merck, Darmstadt, Germany), and polymerized at –20°C for 48 hours. The blocks were coded to allow blind quantification and were processed for sectioning in a Polycut E microtome (Leica, Wetzlar, Germany). Serial 4 µm-thick sections were cut horizontally, ie, perpendicularly to the molar root axis. The sections were sequentially stained with toluidine blue (pH 3.8) or processed for enzymohistochemistry (tartrate-resistant acid phosphatase [TRAP] and alkaline phosphatase, [ALP]) or immunohistochemistry (ED1, monocyte, macrophage, and osteoclast marker). TRAP, a marker of preosteoclasts and osteoclasts in the bone environment, was detected using hexazotised pararosanilin and naphthol ASTR phosphate (Sigma). Nonosteoclastic acid phosphatase activity was inhibited by 50 mmol/L tartaric acid. Sections were lightly counterstained with toluidine blue (pH 3.8). ALP was detected by incubating the sections with naphthol ASTR phosphate and fast blue RR (pH 9). ALP was used to reveal osteogenic cells.

For immunohistochemistry, sections were incubated overnight with a mouse monoclonal antibody directed against ED1 (MAB1435, Chemicon, Temecula, CA), 1:100, and then with a secondary biotinylated antibody (horse anti-mouse IgG; Vector, Burlingame, CA; 1:200, 90 minutes). After treatment with 3% hydrogen peroxide for 10 minutes and avidin–biotin peroxidase complex (ABC Vectastain Kit, Vector) for 1 hour, the chromogen 3,3'-diaminobenzidine tetrahydrochloride (Sigma) was added. Negative controls were prepared by omitting the primary antibody, by replacing the primary antibody with horse non-immune serum at the same dilution or by using an irrelevant secondary antibody.

In Vitro Osteoclastogenesis

To test the effect of histamine deficiency, we used cells from mice with a targeted disruption of the HDC gene. HDC–/– mice were generated as previously described20 and backcrossed onto the C57BL/6 background for more than six generations at the time of experiment and were therefore considered as being in pure C57BL/6 background. Control wild-type C57Bl6/J mice were purchased from the Charles River Laboratories (L’Arbresle, France).

In vitro osteoclastogenesis was performed according to Kim et al, with modifications.21 Briefly, spleen cells were isolated from 6 to 10-week-old wild-type and HDC–/– mice and cell suspension was obtained using a 70-µm nylon mesh cell strainer. After a red blood cell lysis step, cells were washed twice with {alpha}-MEM, and seeded in 0.4 ml {alpha}-MEM with 10% fetal bovine serum in 8-well plates (1.106 cells/cm2). After attachment overnight, cultures were fed every 3 days by replacing 50% of the medium with fresh medium for 14 days, and supplemented with M-CSF (25 ng/ml) in the presence or absence of RANKL (50 ng/ml). In this system, splenocytes proliferate, differentiate into mononuclear TRAP-positive cells, and fuse to form osteoclast-like cells (OCLs) after 2 weeks. For resorption assays, spleen cells (1.105 cells/well) were plated on dentin slices as previously described.21 Briefly, the slices were cleaned in ethanol and each slice was then placed into 48-well plates. Spleen cells were added into each well, cultured in {alpha}-MEM with 10% fetal bovine serum with M-CSF (25 ng/ml) and RANKL (50 ng/ml) and maintained for 14 days. The slices were then recovered, cleaned by ultrasonication and stained with toluidine blue. The number of resorption lacunae (pits) on each slice was counted under a light microscope.

Cell cultures were incubated with increasing concentrations of histamine for 2 hours or with H1R antagonist mepyramine (10–6 M/L), H2R antagonist famotidine (10–6 M/L), H3R antagonist ciproxifan (10–5 M/L), an H4R antagonist JNJ 7777120 (10–6 M/L), or {alpha}- fluoromethylhistidine (10–6 M/L), a selective HDC inhibitor. Concentrations used here were chosen to ensure a specific effect and avoid effects not attributable to HR binding; treatment protocols, concentrations, frequency, and duration are similar to those of previous reports.14,22,23 After 2 hours, the medium was supplemented with 10% fetal calf serum, and changed after 3 days. Histamine content in the medium was quantified by an automated continuous flow spectrofluorometric technique.24

At the end of the culture period, the cells were washed with PBS, fixed with 4% paraformaldehyde, stained for TRAP and nuclear counterstained with methyl green solution. TRAP-positive multinucleated (number of nuclei >3) cells were considered OCLs and were counted under the microscope.

Calvarial Osteoblast Primary Culture

Primary osteoblasts were enzymatically isolated from calvariae of neonatal (2- to 3-day-old) wild-type mice.25 Briefly, calvariae were dissected aseptically and sequentially digested for 70 minutes in a PBS collagenase solution containing 0.2% collagenase IV (Sigma) and 0.01% deoxyribonuclease (Sigma) at 37°C. Calvarial cells were collected by centrifugation, washed and plated at 1.106 cells/25 cm2 flasks. Cells were expanded for 5 days in {alpha}MEM supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were harvested with trypsin/EDTA (Invitrogen) and plated at a density of 1.105 cells/cm2 in the differentiation medium (50 µmol/L ascorbic acid with or without 1.10–8 mol/L 1,25-[OH]2VitD3) and treated with histamine as described above.

RNA Extraction and Real-Time Quantitative PCR

Total RNAs from primary cell lysate (spleen cells derived osteoclasts and osteoblasts) were isolated at different time points using trizol reagent (Invitrogen) and cleaned using an RNAeasy minikit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. The RT reaction was performed using the Reverse-ITmax RTase Blend (Abgene, Courtaboeuf, France). Quantitative real-time PCR expression analysis was performed using a light cycler 7 Roche, ABsolute SYBR Green Capillary Mix (ABgene). Aldolase A and 18S were used for normalization. Sets of primers were designed from the online mouse library probes of Roche Diagnostics for specific detection of the following genes: Aldolase A, 18S, RANKL, OPG, H1R, H2R, H3R, H4R, HDC, TRAP, and calcitonin receptor.

Statistical Significance

In vitro experiments were repeated at least three times independently. Results were expressed as mean ± SE (SEM). Statistical analysis was performed by Statview analysis program using two-way analysis of variance. Where significant overall differences were detected by analysis of variance, Fisher’s two-tailed unpaired t-test was used to compare differences between treatments. P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histamine Modulates Monocyte Recruitment and Osteoclast Differentiation in Vivo

Circulating monocytes are potential osteoclast precursors and therefore we first assessed whether monocyte, identified as ED1+ cells, recruitment close to the bone surface was histamine-dependent. In this aim, we used successively activatory and inhibitory approaches. Injection of histamine or c48/80 (to induce degranulation of residing mast cells), strongly increased monocyte recruitment (+103%, P < 0.01 and + 86%, P < 0.04, respectively, Figure 1A ). Conversely, using H1R and H2R antagonists, we found that their recruitment was reduced compared with animals treated with VEH (Figure 1A) . H2R and H1R antagonist decreased ED1+ cells dramatically (–72.4%, P < 0.003 and –59.8%, P < 0.02 respectively). Bone resorption was directly correlated with the changes in monocyte recruitment. Indeed, 4 days after induction, osteoclast numbers were strongly increased with local histamine and C48/80 (+67.8%, P < 0.0001 and +41.7%, P < 0.0005, respectively) (Figure 1, B–D) . They were reduced with H1R antagonist (–21.6%, P < 0.01) and with H2R antagonist (–42.2%, P < 0.005) (Figure 1, B, C, E and F) given immediately after extraction (early treatment). Beside histamine impact on monocyte recruitment and subsequently on the number of differentiated osteoclast, we also looked for a direct effect of histamine on osteoclast differentiation and activity in vivo, independent of the recruitment of monocytes. Histamine receptor antagonists were thus injected 24 hours after extraction (delayed treatment), ie, after the entry of monocytes close to the bone surface was completed. In this situation, the number of osteoclasts was strongly decreased with anti-H1R treatment and moderately after the anti-H2R treatment (–51.2%, P < 0.0005 and –18.9%, P < 0.005, respectively) (Figure 1, B, G, and H) . Local injection of histamine without induction of the resorption wave (extraction of the maxillary molars) did not lead to resorption of the bone surface (data not shown).


Figure 1
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  		Figure 1. Histamine modulates monocyte recruitment and osteoclast differentiation in vivo. TRAP periosteal osteoclasts differentiate at 4 days postinduction from mononucleated ED1+ precursors entering the site 12 hours after induction. Rats were treated locally with either histamine, compound 48/80 or saline (VEH). Rats were also treated systemically with either a H1R antagonist, a H2R antagonist or saline (VEH) injected immediately after induction (early treatment) or 24 hours later, ie, after ED1+ cell entry (delayed treatment). A: Changes in ED1+ cell counts. B: Variations in TRAP+ cell counts. C to H: TRAP+ osteoclasts resorbing the cortical bone in the different treatment groups. Data are shown as means ± SE. *P < 0.05; **P < 0.005, different from VEH group. ***P < 0.05, different from H1R antagonist group. Scale bar = 100 µm.

 
Histamine Promotes Osteoclasts Differentiation and Function in Spleen Cell Cultures

To confirm the in vivo data on histamine effect on osteoclast differentiation, we used spleen cells cultures from HDC–/– and wild-type mice, in which osteoclast progenitors were induced to differentiate with RANK-L and M-CSF in absence of stromal/osteoblast cells. Fewer osteoclast progenitors differentiated into OCLs in the HDC–/– than in the wild-type cultures (–34.4%, P < 0.01) (Figure 2A) . By adding histamine in the culture medium, osteoclast formation was restored in HDC–/– cultures while it was enhanced in wild-type cultures (+37% at 10–6M, P < 0.005). These results were confirmed by inhibiting histamine synthesis with {alpha}-fluoromethylhistidine in wild-type cells that markedly decreased osteoclast differentiation, similar to HDC–/– cells (–29.7%, P < 0.02). A dose–effect relationship was observed between histamine concentration and the number of OCLs (Figure 2B) . In a time-course experiment, we observed that starting the histamine treatment early during cultures markedly increased osteoclast differentiation (Figure 2C) . Next, we assessed the role of histamine on osteoclast activity. Resorption on dentin by OCLs from HDC–/– mice was reduced compared with wild-type cells (–45.5%, P < 0.02) (Figure 2, D and E) . Altogether, spleen cell culture showed that the early stage of osteoclast differentiation and OCL activity were enhanced in presence of increasing amount of histamine.


Figure 2
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  		Figure 2. Histamine promotes differentiation and function of osteoclast in spleen cells cultures. Osteoclastogenesis was assessed in wild-type (WT) and HDC–/– spleen cells cultured with M-CSF and RANKL for 2 weeks. Osteoclast-like cells (OCLs) were defined as TRAP positive cells with three or more nuclei. A: Wild-type and HDC–/– spleen cells were treated with histamine (10–6M) for 2 weeks. B: Histamine dose-dependently affects the number of OCLs. C: The importance of histamine treatment period on OCLs formation was assessed. Spleen cells were treated with histamine (10–6M) for 3, 7, or 14 days (d0 to d3, d0 to d7, and d0 to d14). D and E: OCLs activity was tested for pit formation on dentin slices. The resorbed area was quantified after toluidine blue staining. Data are shown as mean ± SEM. *P < 0.05, **P < 0.005, different from wild-type cells receiving RANKL. ***P < 0.05, different from HDC–/– cells receiving RANKL. n = 4 wells per genotype and condition. The result is representative of three independent experiments. Scale bar = 50 µm.

 
Histamine Is Synthesized in Osteoclasts Precursors

To explain the observed differences between wild-type and HDC–/–, we looked for the production of histamine in spleen cell cultures. We first assessed typical markers of the osteoclast phenotype (calcitonin receptor and TRAP) and confirmed that osteoclast differentiation was associated with increase mRNA expression of these markers (Figure 3, A and B) . We detected HDC mRNA in OCLs and its expression decreased as the cells differentiated (Figure 3C) . Histamine content was then quantified in the medium. We showed that while histamine was totally absent in the fresh medium, it was present at day 7 and day 14 (range of histamine concentration 50/90 ng/ml and 2.5/3.5 ng/ml, approximately 10–7M and 10–8M respectively at day 7 and day 14) (Figure 3D) .


Figure 3
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  		Figure 3. Histamine is synthesized in osteoclasts precursors and its receptors are differentially expressed during osteoclast differentiation. Spleen cells from wild-type mice were cultured with M-CSF and RANKL or M-CSF alone for 2 weeks. Total RNA was isolated from primary cell lysate at different time points. Relative expression levels of TRAP, calcitonin receptor, HDC, H1R, and H2R were determined by real-time PCR at days 3, 7, 10 and 14 (A, B, C, E and F). Results are presented as mean ± SEM of triplicates. *P < 0.05, different from day 3. Histamine content was quantified in the cell culture medium by an automated continuous flow spectrofluorometric technique at days 0, 7, and 14. D: For this particular experiment, the medium was replaced completely at each medium change. The functional role of the histamine receptors on osteoclastogenesis was evaluated using selective HR antagonists in cell cultures. Cells were treated for a determined period (d0 to d7, d7 to d14, or d0 to d14) with antagonists of H1R (10–6M), H2R (10–6M), H3R (10–5M), H4R (10–6M), or saline (VEH). OCLs were quantified after 2 weeks in culture G: Results are presented as mean ± SEM. **P < 0.05, different from VEH cells. n = 4 wells per condition. The result is representative of three independent experiments.

 
Histamine Receptors on Osteoclasts Are Differentially Expressed during Differentiation

Since we detected locally produced histamine, we investigated the expression of the different histamine receptors that could mediate the effect of histamine in OCLs. H1R mRNA expression remained stable during osteoclastogenesis, whereas H2R mRNA expression was maximal at the early stages of osteoclast differentiation and decreased continuously after day 7 of culture (Figure 3, E and F) . Expression profile induced by M-CSF plus RANKL was slightly different compared with M-CSF alone. H2R was notably less decreased in the presence of RANKL in the culture at day 10. H3R mRNA was not detected in these cultures. H4R mRNA expression was low and inconstantly detected. When detected, H4R increased during the culture period, reaching its maximal level on day 7 (data not shown).

To assess the functional role of these receptors on osteoclastogenesis, we next evaluated the effects of selective HR antagonists in these cultures. When spleen cells were treated with the H1R or H2R antagonists during the whole culture period (d0-d14, Figure 3G ), we observed a significant decrease in osteoclast formation (–36.5%, P < 0.001 and –12.9% P < 0.001 respectively), whereas H3R and H4R antagonists failed to show significant effect. To confirm these findings, we replaced mepyramine with pyrilamine (10–5M, H1R antagonist) and famotidine with cimetidine (10–6M, H2R antagonist) and observed similar results (data not shown). In an additional time course experiment (d0-d7 and d7-d14, Figure 3G ), H1R antagonist inhibited significantly OCLs formation whether cells were treated during the first or the second period of culture. On the contrary, H2R antagonist was only effective when used during the first period.

Histamine Modulates RANKL/OPG Ratio in Osteoblasts

Osteoblasts play an essential role in osteoclastogenesis through the production of RANKL and OPG. The effect of histamine on RANKL and OPG mRNA expression in wild-type primary osteoblasts was investigated in the presence or absence of 1,25-(OH)2VitD3 at various times up to 14 days postconfluence. When histamine (10–6M) was added in cultures with 1,25-(OH)2VitD3, the expression of RANKL mRNA in immature osteoblasts was enhanced compared with cultures containing 1,25-(OH)2VitD3 alone (Figure 4A) . In contrast, OPG mRNA expression was not modified by histamine in presence of 1,25-(OH)2VitD3 (Figure 4B) . Consequently, the RANKL/OPG ratio was significantly increased by histamine in the presence of 1,25-(OH)2VitD3 (Figure 4C) . In the absence of 1,25-(OH)2VitD3, RANKL and OPG mRNA levels were not affected by histamine (data not shown).


Figure 4
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  		Figure 4. Histamine modulates RANKL/OPG ratio in osteoblasts, and its receptors are differentially expressed during osteoblast differentiation. Primary calvarial osteoblasts were cultured with ascorbic acid (50 µmol/L) for 2 weeks in the presence or absence of 1,25-(OH)2VitD3 (10–8M) and histamine (10–6M). Total mRNAs from primary cell lysates were isolated at different time points. Relative expression levels of RANKL, OPG, H1R, and H2R were determined by real-time PCR at days 0, 3, 7, and 14 in culture after induction (A, B, D, and E). Based on the expression levels, RANKL/OPG ratio was calculated for all time points (C). Results are presented as mean ± SEM of triplicates. *P < 0.05, different from the cells treated with ascorbic acid and 1,25-(OH)2VitD3; **P < 0.05, different from the cells treated with ascorbic acid alone. The result is representative of three independent experiments.

 
Histamine Receptors on Osteoblasts Are Differentially Expressed during Differentiation

The observed effect of histamine on the RANKL/OPG ratio prompted us to look for HR expression in the osteoblast lineage, in the presence or absence of 1,25-(OH)2VitD3. H1R and H2R were expressed in primary osteoblasts and their expression appeared to vary according to the differentiation stage (Figure 4, D and E) ; in contrast, H3R and H4R mRNA were not detected. In the absence of 1,25-(OH)2VitD3, H1R mRNA was preferentially expressed in immature osteoblasts, instead H2R expression was strongly expressed in mature osteoblasts. Treatment with 1,25-(OH)2VitD3 abolished the switch of histamine receptors from H1R to H2R during osteoblast differentiation, it enhanced H1R expression in immature osteoblasts and suppressed H2R expression in mature osteoblasts (Figure 4, D and E) . The osteoblasts did not express HDC mRNA or protein in cultures or in histological material, and histamine was not detected in the medium (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We provide here in vivo and in vitro evidence that histamine has a multidirectional action on osteoclastic resorption as it modulates osteoclast precursor recruitment and differentiation. Several cell types are involved and histamine appears to be a critical actor in the interplay between osteoclasts and osteoblasts.

Osteoclasts are derived from common osteoclast/monocyte precursors that are generated in the bone marrow and travel to peripheral tissues through the bloodstream.1 The precursors reach the bone surface after leaving the circulation, transmigrating through endothelial cells. In our in vivo model, monocyte recruitment is shortly preceded by an accumulation and degranulation of mast cells located close to the vessels and histamine release.7,26 We demonstrated in vivo that histamine contained in the mast cell granules triggers precursors recruitment by locally injecting either histamine or c48/80 that induced massive mast cell degranulation. Both agents strongly increased precursors exit from circulation and, subsequently, resorbing osteoclasts 4 days later. On the contrary, histamine receptors antagonists resulted in a reduction in the pool of precursors available and a deficient resorption. In this process of cell recruitment, both H1R and H2R are implicated. These observations agree with the known implication of mast cell degranulation and histamine release in the recruitment of inflammatory cells, including monocytes/macrophages, through up regulation of adhesion molecules on endothelial cells.27

As stated above, increased precursor recruitment resulted in enhanced osteoclastogenesis. It was not clear at that point whether histamine also directly influenced osteoclast differentiation, independently to its role on recruitment. To test this hypothesis, we started the inhibition of histamine receptors after precursor recruitment (delayed treatments) to assess in vivo the effect of histamine blocking on the differentiation of monocyte into osteoclasts. This resulted in a reduction in differentiated osteoclasts, in agreement with our in vitro results obtained with a model of osteoclastogenesis devoid of stromal cells. Indeed, the time-course experiment showed that histamine significantly increases the differentiation of immature proliferating osteoclast precursors. The concentration of histamine measured in the medium of wild-type cell cultures was able to increase osteoclastogenesis when tested on HDC–/– cultures, suggesting that an autocrine process regulates osteoclast precursor differentiation. In fact, this histamine release explains the different outcome in OCL differentiation between HDC–/– and wild-type cultures. A similar mechanism was shown during human monocyte/macrophage differentiation. M-CSF stimulates histamine production while the inhibition of histamine production and function down-regulates the expression of differentiation markers on monocytes28 ; this mechanism operates in macrophage differentiation and function in atherosclerosis.29 Interestingly, the HDC gene is up-regulated in circulating monocytes of patients with low bone mineral density compared with subjects with high bone mineral density,30 strongly suggesting that histamine synthesis by the monocyte lineage is implicated in excessive bone resorption. Altogether, these data establish a direct action of histamine on differentiation and activity through an autocrine mechanism. The release of histamine from other local sources (eg, mast cells, basophils, hematopoietic precursors, and possibly endothelial cells) may also intervene in a paracrine manner (Figure 5) . Systemic mastocytosis, which involves excessive degranulation of mast cells and increased histamine availability close to endosteal bone surfaces, can lead to severe osteoporosis.8,9 Biopsies from patients with systemic mastocytosis shows evidence of increased cortical and trabecular bone turnover in regions of mast cell accumulation.31 Using other in vitro models (bone marrow cells or co-culture of osteoblasts and bone marrow), previous studies already showed that histamine promotes osteoclast differentiation12,13,14,15 and it was suggested that this effect of histamine could be explained by the increased RANKL expression in osteoblasts.


Figure 5
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  		Figure 5. Histamine as an autocrine and paracrine agent regulating osteoclast differentiation. Histamine synthesis by osteoclast/monocyte precursors promotes osteoclast formation directly through autocrine/paracrine action on the precursors and indirectly through increased osteoblasts secretion of RANKL. The release of histamine from other local sources (eg, mast cells, basophils) may also intervene in a paracrine manner during normal and pathological situations in the periosteum and the endosteum.

 
In this study, using primary osteoblasts we found that histamine targets osteoblasts and modulates RANKL/OPG ratio and confirmed that RANKL was up- regulated14,15 ; in contrast, OPG expression was not affected by histamine, so that RANKL/OPG ratio shifted in favor of osteoclastogenesis. By locally secreting histamine, osteoclast precursors probably induce an increase in RANKL/OPG ratio by the osteoblasts, which in turn enhances osteoclast differentiation and activity, thus providing a rare example of communication in this direction.4

The effects of histamine depend on the subtypes of histamine receptors present on the target cells6 ; we observed that histamine receptor expression on osteoclasts varied with the differentiation stage. In vivo, depending on the timing of treatment, the ability of HR antagonists to inhibit osteoclast formation varied, suggesting that H1R and H2R have different roles in this process and are differentially expressed. In vitro, we showed that osteoclast precursors expressed both H1R and H2R, whereas mature OCLs mainly expressed H1R. Accordingly, monocytes preferentially express H2R rather than H1R while the differentiation into macrophages of human peripheral blood monocytes and the U937 cell line is accompanied by enhancement of H1R expression.32 As histamine dose- and time-dependently prevents apoptosis in monocytes through H2R,33 this could expand the pool of cells available for differentiation in the osteoclast pathway. In vivo, the involvement of H2R in bone resorption was also evidenced as their antagonists partially inhibited bone loss in models of osteoporosis,34 arthritis,12 and periodontitis.35 In bone marrow cultures, H2R antagonists partially inhibited the histamine-dependent increase in osteoclast formation.12,15 However, our data suggest that both H1R and H2R are involved in bone resorption and that H1R expression on multinucleated osteoclasts also plays a central role. In vivo, delayed treatment with H1R antagonist (ie, starting after precursor recruitment) had a stronger inhibitory effect on osteoclast differentiation than with H2R antagonist. The same was observed in vitro since the H1R antagonist was the only antagonist effective on the late stage of OCL culture. Clinical studies support histamine and H1R involvement in bone resorption. In a study investigating whether pollen-allergy can affect bone mass and fractures in postmenopausal women, it was shown that H1R antagonists may compensate for the negative effect of allergy on bone fracture risk.36 The use of an H1-antihistamine/mast cell stabilizer in a patient with systemic mastocytosis and extensive skeletal involvement, including elevated parameters of bone resorption, improved the bone parameters.37

We confirmed that osteoblasts also express H1R and H2R14,15 and showed that in primary osteoblasts their expression varied with the differentiation stage. Interestingly, H1R was preferentially expressed in immature cells, which support proliferation and differentiation of osteoclasts through secretion of cytokines like RANKL and M-CSF,38 whereas H2R was expressed in mature cells. The treatment of the cultures with 1,25-(OH)2VitD3, enhanced H1R expression in immature osteoblasts and markedly decreased H2R expression in mature osteoblasts. In fact, H1R is a downstream target for the vitamin D receptor in stromal cells during osteogenic differentiation.39 By regulating the subtype and distribution of histamine receptors on osteoblast and increasing H1R on immature osteoblast, 1,25-(OH)2VitD3 modifies the effects of histamine on the cell and could explain why RANKL/OPG ratio is not modified by histamine in the absence of 1,25-(OH)2VitD3. It was previously shown that the increase in RANKL expression induced by histamine was H1R-dependent.14,15 Interestingly, Fitzpatrick et al observed an increased vitamin D3 synthesis in HDC–/– mice and that a vitamin D-deficient diet prevented the increase in bone mass observed in these mice.13

In summary, our study provides evidence for histamine involvement in osteoclast differentiation and for specific roles of H1R and H2R. We showed that histamine promotes osteoclast differentiation directly through autocrine/paracrine action on the precursors and indirectly through an increased RANKL/OPG expression ratio by osteoblasts. H1R and H2R distribution on bone cells is highly controlled; modulation of expression of H2R on osteoclast precursors and up-regulation of H1R on osteoblast by 1,25-(OH)2VitD3 are key events by which histamine promotes osteoclastogenesis. The clinical relevance of our observations is highlighted in inflammatory bone diseases with excessive resorption (eg, osteoporosis, arthritis, systemic mastocytosis, or periodontitis) in which mast cells and other histamine-forming cells are stimulated to release histamine close to bone surfaces. Our results suggest that anti-histaminic compounds or HDC inhibitors may be used to inhibit osteoclastic activity and bone resorption in these pathological conditions.


   Footnotes
 
Address reprint requests to Jean-Louis Saffar, EA 2496, Université Paris Descartes, Faculté de Chirurgie Dentaire, 1 rue Maurice Arnoux, 92120 Montrouge, France. E-mail: jean-louis.saffar{at}univ-paris5.fr

Supported by the Institut Francais pour la Recherche en Odontologie.

Accepted for publication December 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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