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1 Laboratoire sur la réparation et les remodelages oro-faciaux, EA 2496, Université Paris Descartes, Faculté de Chirurgie Dentaire, 92120 Montrouge, France
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(Received 9 January 2006;
accepted after revision 13 February 2006; first published online 2 March 2006)
Corresponding author J. L. Saffar: Laboratoire sur la réparation et les remodelages oro-faciaux, Faculté de Chirurgie Dentaire, Université Paris Descartes, 1 rue Maurice Arnoux, 92120 Montrouge, France. Email: jean-louis.saffar{at}univ-paris5.fr
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Introduction |
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At least two cell types are probable sources of histamine in the bone marrow of OVX rats. Mast cells (MCs), the main cell type synthesizing and storing histamine, release histamine upon activation and/or degranulation; MC numbers increase in the femur marrow of OVX rats (Lesclous & Saffar, 1999) and are diminished by H2R antagonism (Lesclous et al. 2002, 2004). Other histamine-producing cells also populate the bone marrow. Schneider et al. (1993) described a haematopoietic population that constitutively synthesizes histamine and belongs to an immature population at an intermediate stage of differentiation between pluripotent cells and lineage-committed precursors. These cells do not store histamine, which is therefore secreted as soon as it is synthesized (Schneider et al. 2002). Their numbers increase after OVX and diminish during H2R antagonism. Thus, the two populations are both targeted, directly or indirectly, by oestrogen depletion (Lesclous et al. 2004). Interestingly, Harnish et al. (2004) showed that oestrogen inhibits in vitro MC degranulation and release of inflammatory cytokines, such as tumour necrosis factor-
(TNF-) and interleukin-6 (IL-6). Tumour necrosis factor- and IL-6 release inhibition was also obtained with 17ß-oestradiol in vitro in human MCs (Kim et al. 2001). It is thus likely that oestogen depletion directly impacts on MCs, resulting in their activation and in subsequent release of histamine and inflammatory cytokines.
Our previous studies showed the preventive effect of short (14 days) anti-H2R treatments on OVX in bone loss (Lesclous et al. 2002, 2004); however, to better understand this effect, H2R antagonism should be evaluated over a longer period and in another anatomical site responding differently to oestrogen depletion relative to the appendicular skeleton. In contrast to the rapid bone loss affecting long bones (Wronski et al. 1988), vertebral bone loss remains moderate during the first 6 months after OVX, before increasing markedly (Wronski et al. 1989; Liu & Kalu, 1990; Bagi et al. 1994). Despite its delayed reactivity, the rat vertebra is a site of interest because of its relevance to human osteoporosis, although in postmenopausal women collapse of the spine occurs earlier than femoral fracture. This may be due to the different weight-bearing functions of long bones and the spine, in that long bones support higher mechanical loads than the vertebral column, particularly in quadrupeds, and this may contribute to a more rapid response to the loss of ovarian function (Wronski et al. 1999). In this 6 month study, we examined the bone-protective effect of H2R antagonism in the distal femur and the lumbar spine.
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Methods |
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Thirty-two 90-day-old female Wistar rats (Iffa-Credo, l'Arbresle, France) weighing 260 ± 20 g were housed, one per cage, in a room with a 12 h–12 h light–dark cycle. They were fed 15 g day–1 of a rodent diet containing 0.3% calcium (AO4, UAR, Villemoisson, France), as recommended by Sims et al. (1996) to prevent weight gain in ovariectomized rats (Kalu et al. 1989). Water was supplied ad libitum. The study complied with European Union recommendations on laboratory animal care.
Bilateral ovariectomy was performed in 16 rats. The animals were anaesthetized for surgery with an 8% chloral hydrate solution (Prolabo, Fontenay France) via the I.P. route. The ovaries of the other rats were surgically exteriorized and reinserted into the abdomen (sham-operated controls). Famotidine (Sigma, St Louis, MO, USA) dissolved in saline was injected intramuscularly (I.M.) into eight sham and eight OVX rats (10 mg kg–1 day–1). Famotidine, a reference H2 antagonist, was used in place of cimetidine, which we used in our former study (Lesclous et al. 2002), because it is more potent. The two agents differ in their chemical structure (famotidine has a thiazole ring while cimetidine, like histamine, contains an imidazole ring; the grafted radicals are also different) and in their pharmacokinetics (Feldman & Burton, 1990). The other animals received daily I.M. injections of saline. Calcein (30 mg kg–1) and demeclocycline (30 mg kg–1) (both from Sigma) were injected intraperitonealy, respectively, 7 days and 1 day before killing. The rats were weighed at the start of the experiment and monthly thereafter. The animals were anaesthetized with an 8% chloral hydrate solution (Prolabo) via the I.P. route and killed by cardiac exsanguination.
Sample processing
The right femur, the fourth lumbar vertebra and the uterus were removed. The distal part of the femur and the whole vertebra were fixed in cold 70% ethanol. After dehydration the bones were embedded without demineralization in methyl methacrylate (Merck, Darmstadt, Germany). Longitudinal sections (4 and 8 µm thick) were cut using a Polycut E microtome (Leica, Wetzlar, Germany) and stained with Toluidine Blue (pH 3.8) or von Kossa technique; the vertebrae were also processed for enzyme or immunohistochemistry to detect tartrate-resistant acid phosphatase (TRAP) and histamine, respectively. The uterus was weighed to confirm successful OVX (Wang et al. 2001). Fluorochrome-based indices of bone formation were measured in unstained 8-µm-thick sections.
TRAP was detected using Fast Red TR salt (Sigma-Aldrich, Saint Quentin Fallavier, France) and naphthol ASTR phosphate (Sigma). Non-osteoclastic acid phosphatase activity was inhibited with 50 mmol l–1L(+)-tartric acid added to the substrate solution. Histamine was revealed with a rabbit polyclonal antibody (H-7403, Sigma). The sections were incubated overnight with 0.1 M PBS supplemented with 0.05% Tween (Sigma) and 1% bovine serum albumin (Euromedex, Souffelweyersheim, France) and the primary antibody (1:100) at 4°C in a moist chamber. The sections were then incubated with biotinylated goat antirabbit IgG (Vector, Burlingame, CA, USA) for 90 min at room temperature. They were then treated with 3% hydrogen peroxide (for 10 min), and an avidin–biotin peroxidase complex (ABC Vectastain kit, Vector) for 60 min. Phoshate-buffered saline (0.1 M) was used for washing steps between incubations. Diaminobenzidine tetrahydrochloride (Sigma) was used as chromogen. Negative controls were prepared by omitting the primary antibody and by using an irrelevant secondary antibody (horse antimouse IgG instead of goat antirabbit IgG).
Morphometry
Morphometry was performed on the whole secondary spongiosa of the femoral metaphysis (9 mm2 per section on average) as previously described (Lesclous et al. 2002), on the secondary spongiosa of the vertebral body at distances greater than 1 mm from the cranial and caudal growth plates (6 mm2 on average) and on the cortices by using a semi-automatic image analyser. The following parameters were recorded in accordance with the american society for bone and mineral research (ASBMR) nomenclature committee (Parfitt et al. 1987): the trabecular bone volume (TV/BV, expressed as a percentage of the bone volume), the osteoid surface (OS/BS, expressed as a percentage of the trabecular surface), the resorption surface (Oc.S/BS, expressed as a percentage of the trabecular surface), the number of osteoclasts (N.Oc/BPm, expressed as cells per millimetre of trabecular bone surface), and the average length of the contact zone of osteoclasts with the bone surface (Oc.Pm/N.Oc), an index of osteoclast activity (McMillan et al. 1989) obtained by dividing the total resorption surface (in millimetres) by the number of osteoclasts. In the vertebral samples, we also counted the trabecular number (Tb.N), the mean trabecular thickness (expressed in microns, Tb.Th), the trabecular separation (expressed in microns, Tb.Sp), and the mean cortical bone thickness (expressed in microns, C.Th). Along the vertebral trabeculae and the endocortical surface, the mineralizing surface (MS/BS), the single labelling surface (sLS/BS), the double labelling surface (dLS/BS), the mineral apposition rate (MAR, in microns per day) and the bone formation rate (BFR/BS) were quantified. MS/BS, sLS/BS, dLS/BS and BFR/BS were expressed as a percentage of the bone surface. TRAP-positive preosteoclasts (Fig. 1A) and MC and non-MC histamine-positive cells (Fig. 1B) were also counted; they were expressed as cells per millmetre squared of bone marrow area.
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Data were compared using non-parametric tests (Kruskal–Wallis test followed, if significant, by group comparisons with the Mann–Whitney U test). Differences were considered significant if P
0.05. Results are given as means ±S.E.M.
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Results |
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All the animals gained weight but the weight change was larger in the two OVX groups (P < 0.001). Uterine weight fell in the OVX animals, confirming successful OVX (P < 0.001 versus controls). Famotidine had no effect on these parameters in the sham or OVX rats.
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The trabecular bone volume fell markedly in the OVX rats (–70%; P < 0.0001 versus the corresponding controls). Famotidine treatment did not prevent bone loss in OVX rats (Fig. 2A–C). No difference in indices of resorption or formation was found whatever the treatment of OVX animals. Treatment had no influence on these parameters in sham-operated animals. Cortical thickness was stable (0.62 mm in all the groups). Owing to complete disorganization of the trabecular network, architecture parameters (Tb.N, Tb.Th, Tb.S) and dynamic formation ones (sLS/BS, dLS/BS, MS/BS, BFR/BS and MAR) were not quantified.
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The trabecular bone volume (TB/BV) fell by 40% in the control OVX rats and by 30% in the famotidine-treated OVX rats (P < 0.001 and P < 0.01 versus the corresponding controls, respectively; Fig. 2D–F; Table 3), so that the difference between the OVX groups was significant (P < 0.05). The reduction in TB/BV was related to reductions in trabecula numbers (–23%, P < 0.01) and thickness (–11%, P < 0.01), and to a 40% increase in trabecular separation (P < 0.01). Famotidine significantly attenuated the impact of OVX on these parameters. Ovariectomy reduced cortical thickness by 13% (P < 0.05 versus the corresponding controls), while famotidine maintained it close to the control value (Table 3). Overall, famotidine treatment partly protected the vertebral architecture from the deleterious effect of oestrogen deprivation. Famotidine had no effect in the sham-operated group.
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As previously reported for the femur (Lesclous et al. 2004), the bone marrow contained two histamine-positive cell populations with distinctive morphological features. Mast cells were sparse, large and strongly immunostained for histamine. Smaller cells with a weak and diffuse histamine immunostaining, which were more numerous than the MCs, formed the other population (Fig. 4). Ovariectomy increased the numbers of MC and non-MC histamine-postitive cells by 250 and 80%, respectively (P < 0.001 versus the corresponding controls). The famotidine treatment strongly attenuated these changes; compared with the corresponding sham-operated animals, MCs were only increased by 20% and non-MC histamine-positive cells by 18% (P < 0.05), so that the two OVX groups differed markedly (P < 0.001 and P < 0.01, respectively). Neither histamine-positive population was affected by famotidine treatment in the sham-operated animals (Fig. 5).
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Discussion |
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In the vertebra, the protection provided by famotidine treatment in terms of trabecular bone mass averaged 21% 6 months after OVX, which is close to the value previously found after short-term famotidine treatment in the femur (Lesclous et al. 2004). Accordingly, famotidine treatment reduced the recruitment of TRAP-positive preosteoclasts. This resulted in a certain stability of the bone architecture. As already reported (Okimoto et al. 1998; Li et al. 1999), OVX reduced vertebral cortical thickness owing to extensive endocortical resorption, a feature not seen in the femur despite the extensive trabecular bone loss. This discrepancy may explain why the risk of vertebral collapse is increased in postmenopausal women and occurs earlier than femoral fracture, the weight-bearing nature of the femur possibly protecting it from cortex thinning. The famotidine regimen maintained this parameter close to control values. H2 receptor blockade did not affect bone formation and markedly attenuated the increase in MC and non-MC histamine-producing cell numbers in the vertebra as previously seen in the femur (Lesclous et al. 2004).
The fading of the inhibitory effect of famotidine on femoral bone loss may be due to escape from inhibition, as reported for calcitonin (Wada et al. 1996). However, the observation that H2R inhibition was effective in the vertebra argues against this possibility. More likely, the cytokine and receptor network mediating the early burst of osteoclastic resorption differs from that occurring during the late phase of resorption. According to Dvornyk et al. (2003), the development of osteoporosis is a multistage process, in which each stage is controlled by different sets of genes and mediators. This view of histamine as a first-line mediator agrees with the delayed onset of mechanically activated resorption observed in W/Wv mast cell-deficient mice (Silberstein et al. 1991). During the early phase of resorption, IL-1, TNF- and IL-6 are involved in the burst of osteoclasia (Pacifici et al. 1991; Poli et al. 1994; Kimble et al. 1995; Ammann et al. 1997; Lorenzo et al. 1998). Histamine may also be a key player during this phase, since it induces the production of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage (GM-CSF), IL-6 and IL-1 by haematopoietic cells and circulating mononuclear cells via H2R (Tasaka et al. 1993; Vannier & Dinarello, 1994; Mor et al. 1995; Takamatsu & Nakano, 1998) and synergistically enhances the production of IL-6 by TNF- (Li et al. 2001). These interactions are bidirectional, since histamine production is itself upregulated in turn by these cytokines (Schneider et al. 2002). The rapid impact of oestrogen on MC activation and cytokine release (within 3 days in rats; Harnish et al. 2004) suggests that oestrogen depletion may rapidly initiate the release of histamine, IL-6 and TNF- from MCs and commit precursors in the osteoclastic pathway. We have indeed observed that resorption increases as early as 4 days after OVX (Lesclous et al. 2001). Oestrogen depletion may rapidly activate MCs before targeting the activation of other cell lineages involved in osteoclast differentiation, for instance T lymphocytes (Weitzmann & Pacifici, 2005). Indeed, MCs enhance T cell activation through MC-released TNF- (Nakae et al. 2005). Whether the other histamine-producing population is also directly targeted by oestrogen depletion remains to be determined.
In contrast, cytokine production during the late phase of bone loss has received little attention; most available data come from clinical studies of postmenopausal women, in whom circulating levels of IL-1, IL-1ß, IL-6 and TNF- are similar to those in premenopausal women (Khosla et al. 1994; Sahin et al. 2002). However, these data do not preclude the possibility that the bone marrow concentrations or bioactivity of these mediators are elevated.
In HDC–/– animals, the fall in trabecular bone volume reaches about 26% 3 months after OVX (Fitzpatrick et al. 2003), the same order of change (–30%) found in the vertebra of treated animals in our study; however, since no intermediary data on changes in trabecular volume are available, the kinetics of bone loss in these mice is not known. Bone loss may be increasing in the HDC–/– mice, and at later times osteopenia may be similar in HDC–/– and wild-type OVX mice. If histamine is part of the mediator network regulating the early phase of osteoclasia, a histamine-independent late increase in resorption might eliminate the trabecular bone that was initially protected in these mice, as in our rats.
In conclusion, the arrest of resorption inhibition in the femur after 6 months of H2R antagonism suggests that histamine is involved solely in the early phase of bone destruction that follows ovarian suppression. The significant inhibition of resorption in the lumbar spine, a site where bone loss starts later than in the appendicular skeleton, supports this hypothesis. Thus, oestrogen depletion directly or indirectly modulates histamine release in bone marrow, and histamine in turn may induce and/or enhance cytokine production in this organ.
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P < 0.01,