JOURNAL OF BONE AND MINERAL RESEARCH Volume 21, Number 11, 2006 Published online on July 31, 2006; doi: 10.1359/JBMR.060726 © 2006 American Society for Bone and Mineral Research
T Lymphocyte–Deficient Mice Lose Trabecular Bone Mass With Ovariectomy Sun-Kyeong Lee,1 Yuho Kadono,2 Fumihiko Okada,2 Claire Jacquin,3 Boguslawa Koczon-Jaremko,1 Gloria Gronowicz,4 Douglas J Adams,4 Hector L Aguila,3 Yongwon Choi,2 and Joseph A Lorenzo1
ABSTRACT: We examined OVX-induced bone loss in three TLD mouse models. In TLD mice, OVX caused trabecular bone loss equivalent to that of WT. In contrast, cortical bone loss with OVX was variable. We conclude that T lymphocytes do not influence OVX-induced trabecular bone loss. Introduction: We examined ovariectomy (OVX)-induced bone loss in three T lymphocyte–deficient (TLD) mouse models: nude mice, recombination activating gene 2–deficient (RAG2 KO) mice, and ⌻ cell receptor ␣ chain–deficient (TCR␣ KO) mice. Materials and Methods: Bone mass was examined by DXA, CT, and histomorphometry. We also examined the effect of OVX on T lymphocytes in the bone marrow and spleens of wildtype (WT) mice and on in vitro osteoclastogenesis and colony forming unit-granulocyte macrophage (CFU-GM) activity in the bone marrow of WT and nude mice. Results: In WT mice, OVX did not alter T lymphocyte number in the bone marrow but did increase T lymphocytes in the spleen. Comparison of bone mass in nude, RAG2 KO, and TCR␣ KO mice with WT as measured by DXA showed decreased femoral bone mass in nude mice and increased vertebral bone mass in RAG2 KO mice. In TCR␣ KO mice, femoral, tibial, and vertebral bone mass were decreased. In vertebrae and long bones, bone loss with OVX was consistently present in WT mice but variably present in TLD mice as measured by DXA. In contrast, CT and histomorphometry showed similar trabecular bone loss after OVX in all mice. However, femoral cortical bone loss occurred only in WT and RAG2 KO mice. OVX produced similar trabecular bone loss in WT and TCR␣ ⌲⌷ mice and also induced cortical bone loss in both. Histomorphometry showed that TRACP+ area in bones was increased by OVX in femurs from both WT and nude mice as was in vitro osteoclast-like cell formation and CFU-GM activity. Conclusions: These results show that OVX caused similar trabecular bone loss in both WT and TLD mice. The ability of DXA and measurement of cortical bone loss to show OVX-induced effects on bone mass was variable. It seems that T lymphocytes are not critical for OVX-induced trabecular bone loss in these mouse models. J Bone Miner Res 2006;21:1704–1712. Published online on July 31, 2006; doi: 10.1359/JBMR.060726 Key words: T lymphocyte, ovariectomy, osteoclasts
INTRODUCTION
T
LYMPHOCYTES ARE CRITICAL regulators of immune cell function and an important source of signals that regulate bone turnover.(1) The role of T lymphocytes in bone was first shown by the discovery that the conditioned medium (CM) from cultures of human peripheral blood mononuclear cells, which were stimulated with the T cell–specific activator phytohemagglutinin, increased bone resorption in fetal bone explants.(2) This activity was labeled osteoclast
Dr Lorenzo holds consultancies with Amgen and Aastrom. The other authors state that they have no conflicts of interest.
activating factor (OAF). More recently, depletion of T lymphocytes either in vitro(3) or in vivo(4) was shown to regulate osteoclastogenesis that was stimulated in vitro by treatment of depleted bone marrow with stimulators of resorption. It is now known that T lymphocytes produce a variety of cytokines that have pro- and anti-osteoclastogenic potential. Principal among these is RANKL,(5,6) which is a central mediator of osteoclast-mediated bone resorption. In addition, a large number of cytokines are known to influence osteoclastogenesis. Inhibitors of resorption that are produced by T lymphocytes or which regulate T-lymphocyte function include IFN-(7) and -␥(8) and interleukin-4 (IL-4),(9) IL-10,(10) IL-12,(11) IL-13,(12) IL-17,(13)
1 Division of Endocrinology, Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA; 3 Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA; 4Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut, USA. 2
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T CELL–DEFICIENT MICE LOSE BONE MASS WITH OVARIECTOMY and IL-18.(14) In addition, T lymphocytes interact with other immune cells to stimulate the production of additional bone resorption stimuli including IL-1, IL-6, and TNF-␣.(15) Estrogens have a significant ability to regulate bone mass.(16) At menopause, there is an acceleration of the rate that bone is lost in women, which decreases when postmenopausal women receive estrogen replacement therapy.(17) The role that the immune system has in this process has been studied in some detail, and it seems, at least in rodent models, that interactions of immune mediators with bone cells are responsible for some or all of the bone loss that occurs after estrogen withdrawal.(18) Specifically, it was shown that mice deficient in IL-1 type I receptor (IL1R1),(19) IL-6,(20) and TNF-␣(21) did not lose bone mass after ovariectomy (OVX). Furthermore, mice made deficient in IL-7 by injection of a neutralizing antibody also are reported not to lose bone mass after OVX.(22) B-lymphopoiesis is increased by estrogen withdrawal,(23) and it was postulated that interactions of B lymphocytes with bone cells may cause some of the bone loss that occurs after estrogen withdrawal.(24) Recently, T lymphocytes were also implicated in the loss of bone mass that accompanies estrogen withdrawal.(25) Nude mice have a severe deficiency in T lymphocytes because they lack adequate thymic stromal support functions.(26) It was found that nude mice did not lose trabecular bone mass after estrogen withdrawal as measured by pQCT.(25) However, in an earlier study, using histomorphometry, it was reported that there were no difference in the rates of trabecular bone loss after OVX between nude rats and wildtype (WT) control animals.(27) Like nude mice, nude rats have a severe deficiency in T lymphocytes. To attempt to clarify these conflicting results, we examined the ability of nude mice and two additional T lymphocyte–deficient mouse models, T cell receptor ␣ (TCR␣)– deficient mice(28) and recombination activating gene 2 (RAG2)–deficient mice, to lose bone mass after OVX. In our studies, we measured bone mass by DXA, which measures total bone mass, and CT and histomorphometry, which measure cortical and trabecular bone mass separately.
MATERIALS AND METHODS Experimental animals WT, nude (B6.Cg/NTac-Foxnl n u N9), RAG2 KO (B6.129S6-Rag2tm1FwaN12), and TCR␣ KO (B6.129S2Tcra tm1Mom/J) mice in a C57BL/6 background were used for all experiments. Initial WT experiments were performed using C57BL/6 mice from Charles River Laboratories (Wilmington, MA, USA). Nude mice, RAG2 KO, and their WT controls were purchased from Taconic (Hudson, NY, USA). TCR␣ KO mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Animals were housed in the Centers for Laboratory Animal Care at the University of Connecticut Health Center and the University of Pennsylvania School of Medicine. All animal protocols were approved by the animal care committees of the
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University of Connecticut Health Center and the University of Pennsylvania School of Medicine. At 5–6 weeks of age, mice were either sham (SHAM) operated or OVX and killed 4 (nudes and RAG2 KO mice) or 8 weeks (TCR␣ KO mice) later. OVX was confirmed by measurements of uterine weight at the time of death.
Static and dynamic histomorphometry, DXA, and µCT analysis Bone histomorphometry was performed on mouse femurs. Histomorphometric analysis was performed in the Center for Bone Histomorphometry at the University of Connecticut Health Center in a blinded, nonbiased manner using a computerized semiautomated system (Bioquant, Nashville, TN, USA) interfaced with an Optiphot Nikon microscope (Nikon, Melville, NY, USA). The bones from at least 11–14 mice per group were examined. Animals received intraperitoneal injections of 10 mg/kg of calcein 9 days and xylenol orange (90 mg/kg) 2 days before death. For static histomorphometry, the femur from each mouse was removed and fixed in 4% paraformaldehyde in PBS at 4°C, decalcified, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. Quantitation of osteoclasts was performed in paraffinembedded tissues that were stained for TRACP. Osteoclasts were identified as TRACP+ cells that were multinucleated and adjacent to bone. Static histomorphometric analysis of one section from each bone was also performed. The measurements, terminology, and units used for histomorphometric analysis were those recommended by the Nomenclature Committee of the American Society of Bone and Mineral Research. (29) In addition, the percent TRACP+ area in a defined total area was obtained for each sample as previously described.(30) Briefly, all measurements were confined to the secondary spongiosa and restricted to an area between 400 and 2000 m distal to the growth plate–metaphyseal junction of the proximal femur. Cortical measurements were made 4000 m distal to the same growth plate. DXA was used to measure BMD of the femur, tibia, and vertebrae (L1) in WT, nude, RAG2 KO, and TCR␣ KO mice that were either SHAM or OVX (PIXImus bone densitometer with dedicated software for analysis of small animals; Lunar Corp., Madison, WI, USA). The femur, tibia, and vertebrae from each animal were removed and preserved in 70% ethanol until DXA measurement. Conebeam X-ray CT (CT40; Scanco Medical AG, Bassersdorf, Switzerland) was used to quantify the trabecular morphometry within L1 vertebral centra and the metaphyses of the distal femur and proximal tibia, as well as the mid-diaphyseal cortex of femurs and tibia. Serial tomographic images were acquired at 55 kV and 145 A, collecting 1000 projections per rotation at 300-ms integration time. Three-dimensional images were reconstructed using standard convolution back-projection algorithms with Shepp and Logan filtering and rendered within a 12.3-mm field of view at a discrete density of 578,704 voxels/mm3 (isometric 12-m voxels). Threshold segmentation of bone from marrow and soft tissue was performed in conjunction
1706 with a constrained Gaussian filter to reduce noise. Volumetric regions for trabecular analysis were selected within the endosteal borders to include the central 80% of vertebral height and secondary spongiosa of the femur (1 mm from the growth plate and extending 1 mm proximally) and tibia (0.5 mm from the growth plate and extending 0.5 mm distally). Trabecular morphometry was characterized by measuring the bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular number (TbN), and trabecular spacing (TbSp). Cortical cross-sectional area was quantified and averaged for 50 serial mid-diaphyseal cross-sections (600 m) located with respect to growth plates.
In vitro osteoclast-like cell formation Bone marrow cells from WT and nude mice were isolated by a modification of previously published methods.(24,31–38) Briefly, bone marrow cells from the femur, tibia, and humerus were collected into tubes, washed twice with ␣-MEM, and cultured (1.5 × 105 cells/cm2) in ␣-MEM containing 10% heat inactivated FBS (HIFBS) for up to 5 days. Cultures were fed every 3 days with fresh medium. Human RANKL-LZ (30 ng/ml), a gift from Dr William Dougall (Amgen, Seattle, WA, USA) and recombinant murine macrophage-colony stimulating factor (rmM-CSF; 30 ng/ml; R & D Systems, Minneapolis, MN, USA) were added at the beginning of culture and replaced with every medium change. Human RANKL-LZ contains an associated leucine zipper protein to increase its activity. Cells were fixed on day 5 of culture with 2.5% glutaraldehyde in PBS for 30 minutes at room temperature before being stained for TRACP. Enzyme histochemistry for TRACP was performed with a commercial kit (Sigma, St Louis, MO, USA).
Colony-forming unit granulocyte-macrophage assay Bone marrow cells were prepared as described previously.(39) Briefly, cells were suspended in ␣−MEM with 10% HIFBS (Hyclone Laboratories, Logan, UT, USA). Cells were plated on a 35-mm tissue culture dish in 1 ml 1.5% methylcellulose supplemented with 20% HIFBS, 2% BSA (Sigma), and 1.0 ng/ml rmGM-CSF (R & D Systems) as the source of colony-stimulating activity. Assays were performed in sextuplets, and cultures were maintained at 37°C for 6 days. The numbers of colonies (>40 cells) were counted at the end of incubation.
Flow cytometry Labeling of bone marrow and spleen cells for flow cytometric analysis was performed on ice using standard staining procedures in 1× HBSS (Gibco; Invitrogen Corp., Carlsbad, CA, USA) containing 0.01 M HEPES (pH 7.4) and supplemented with 2% FBS. Bone marrow cells from the humerus were collected by flushing into a sterile tube using a 25-gauge needle. The medium and suspended cells were spun down, and the pellet was resuspended in 2 ml of ACK (0.15 M ammonium chloride, 1 M potassium bicarbonate, 0.1 M EDTA) to lyse red blood cells. The cells were washed and filtered before being stained with the appropriate dilutions of directly conjugated antibodies (anti-CD3-FITC; eBioscience, San Diego, CA, USA). Anti-CD3 antibody was used as a specific T-cell marker. This antibody binds to
LEE ET AL. both ␣ and ␥␦ T-cell receptors and was used to identify the total T-lymphocyte population. Dead cells were excluded by their ability to incorporate propidium iodide. The samples were run using a Calibur flow cytometer (BD Biosciences, Mountain View, CA, USA) and analyzed using the CellQuest (BD Biosciences).
Statistical analysis Statistical analysis was performed by Student’s t-test when two groups were compared or by one-way ANOVA and the Bonferroni posthoc test if multiple groups were compared. All experiments were repeated at least twice.
RESULTS In our initial experiments, we examined the effects of estrogen withdrawal on the number of T cells in the bone marrow and spleen of WT C57BL/6 mice that were either SHAM or OVX at 8 weeks of age and killed 4 weeks later (Fig. 1). Previous investigators had found an increase in T-cell number in the bone marrow of mice 4 weeks after OVX.(21,40) However, in contrast to those results, we found that the percentage of T cells in bone marrow, which were identified by their expression of the specific T-cell marker CD3, did not show a statistically significant change; although there seems to be a downward trend with OVX. Additionally, there was also no effect of OVX on the absolute number of T cells in bone marrow. In contrast, in the spleen, we found no effect of OVX on the percentage of T cells but an ∼20% increase in the absolute number of T cells caused by an overall increase in total splenic cells after OVX. To further study the role of T cells in estrogen withdrawal–induced bone loss, we examined WT, athymic nude mice, and RAG2 KO mice that were subjected to SHAM or OVX in vivo. Surgeries were performed at 5–6 weeks of age, and all analyses were carried out 4 weeks after surgery to mimic the experimental design of previous studies, which found differences in bone loss after OVX between WT and nude mice.(25) In these experiments, we first examined whether nude mice lost as much uterine weight as WT mice after OVX, because nude mice have previously been found to have a delayed onset of puberty.(41) This effect seems to result from changes in the estrogen responsiveness of tissues from nude mice rather than changes in estrogen levels.(42) However, it was possible that nude mice never achieve the estrogenic responses of WT mice. Uterine weight is a sensitive measure of estrogen levels in mice, and this value rapidly declines with estrogen withdrawal.(19) We found that, in SHAM animals, there was no significant difference in uterine weight between WT and nude mice (data not shown), and in both WT and nude mice, OVX decreased uterine weights by similar amounts (85–87%). Therefore, it seemed that the estrogenic responsiveness of the uterus in WT and nude mice was similar at the time of death. In addition, in SHAM mice, we observed no differences in uterine weight among WT, RAG2 KO, and TCR␣ KO
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FIG. 2. BMD (g/cm2) was measured in the vertebrae (L1) and femur from WT, nude mice, and RAG2 KO mice that were either SHAM or OVX using DXA. Values represent mean ± SE (n ⳱ 3–12/group). *Significant effect of OVX, p ⱕ 0.05. +Significant difference between nude-SHAM and WT-SHAM mice, p ⱕ 0.01. **Significant difference between RAG2 KO-SHAM and WTSHAM mice, p ⱕ 0.05.
FIG. 1. (A) Percentage and (B) absolute number of CD3+ T cells in the bone marrow and spleen of C57BL/6 mice that were either SHAM or OVX. Values are mean ± SE for six to eight determinations per group. *Significant effect of OVX, p ⱕ 0.05.
mice, and we found similar uterine weight loss with OVX in WT, RAG2 KO, and TCR␣ KO mice at the time of death (data not shown). Using DXA to measure total BMD (Fig. 2), we found that there was no significant difference in mean BMD between WT-SHAM and nude-SHAM bones in the vertebrae, but there was in the femur, where nude mice had an 8% lower value (p < 0.01). RAG2 KO SHAM vertebrae had increased bone mass compared with WT SHAM vertebrae, but this was not true for femurs. WT mice lost significant bone mass (7–9%) with OVX in vertebrae (L1), whereas nude mice and RAG2 KO mice did not. In femurs, OVX decreased BMD in WT and RAG2 KO mice but not in nude mice. To further study the role that T lymphocytes had in OVX-induced bone loss, we examined the effect of OVX on WT and ␣ T cell–deficient mice (TCR␣ ⌲⌷). In these experiments, mice were operated on at 6 weeks of age and
killed 8 weeks later. TCR␣ ⌲⌷ mice lack the T-cell receptor ␣ chain and hence have a block in ␣ T lymphocyte development, which are the majority of T cells in mice. BMD was measured in the vertebrae (L1), femur, and tibia by DXA. The mean BMD of both the femur and tibia was significantly decreased in TCR␣ ⌲⌷ SHAM bones (8–9%) compared with WT SHAM (Fig. 3). In WT, OVX induced a significant loss of BMD (5–11%) in all bones examined (vertebrae, femur, and tibia). In contrast, in TCR␣ ⌲⌷ mice, OVX induced a significant loss of BMD (8–11%) in vertebrae and tibias but not in femurs. To further dissect the mechanisms underlying the differences in bone loss after OVX among WT, nude mice, or RAG2 KO mice, we measured trabecular bone mass by CT (Fig. 4) using the same bones that were measured by DXA in Fig. 2. We found no significant difference in vertebral trabecular bone volume among WT SHAM, nude SHAM, and RAG2 KO SHAM bones. However, in femurs, nude SHAM mice had significantly less (26%) trabecular bone volume than did WT SHAM controls or RAG2 KO SHAM bones. In addition, nude SHAM but not RAG2 KO SHAM mice had significantly decreased trabecular number (14%) and increased trabecular spacing (20%) compared with WT SHAM bones (data not shown). There was no difference in trabecular thickness among bones from WT SHAM, nude SHAM, or RAG2 KO SHAM mice (data not shown). In contrast to the DXA measurements of total bone mass, we found that there were significant effects of OVX on the trabecular bone mass of WT, nude, and RAG2 KO mice, which were similar (Fig. 4). OVX decreased trabecular bone volume (BV/TV) by 21–37% in the vertebrae and 40–45% in femur of WT, nude, and RAG2 KO mice compared with their respective SHAM controls. OVX also decreased trabecular number and increased trabecular spacing in vertebrae and femurs from WT, nude, and RAG2 KO mice by similar amounts (21–32% decrease in femoral TbN, 5–16% decreased in vertebral TbN, 31–50% increase
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FIG. 3. BMD (g/cm2) was measured in the vertebrae (L1), femur, and tibia of WT and TCR␣ KO mice that were either SHAM or OVX using DXA. Values represent mean ± SE (n ⳱ 14–25/ group). *Significant effect of OVX, p ⱕ 0.05. +Significant difference between TCR␣ KO-SHAM and WT-SHAM mice, p ⱕ 0.05.
FIG. 4. Trabecular volume (BV/TV) of vertebrae (L1) and femur and cortical volume of femur measured by microCT collected from WT, nude mice and RAG2 KO mice that were either shamoperated (SHAM) or ovariectomized (OVX). Values represent mean± SEM (n ⳱ 3–12 per group). *, Significant effect of ovariectomy, p ⱕ 0.05. #, Significant effect of ovariectomy, p ⳱ 0.05. +, Significant difference between nude-SHAM and WT-SHAM mice, p ⱕ 0.05. **, Significant difference between RAG2 KOSHAM and WT-SHAM mice, p ⱕ 0.05.
in femoral TbSp, 8–21% increase in vertebral TbSp). In contrast, we found that cortical bone area in the midshaft of the femur was decreased by OVX in WT bones (p ⳱ 0.05) but was not altered in nude mice. Interestingly, cortical bone area was increased in the femurs of RAG2 KO SHAM mice compared with WT SHAM mice, and as with WT mice, OVX induced a loss of cortical bone area in RAG2 KO mice (p ⳱ 0.05). There was no significant difference between the femoral cortical bone mass of WT SHAM and nude SHAM mice. We next used CT (Fig. 5) to measure the effects that OVX had on trabecular and cortical bone mass in WT and TCR␣ ⌲⌷ mice using the same bones that had been previously analyzed by DXA in Fig. 3. Unlike the results in nude mice, there was no significant difference in femoral, tibial, or vertebral trabecular bone volume between WT SHAM and TCR␣ ⌲⌷-SHAM bones. However, there was a trend for BV/TV to be decreased in the vertebrae of TCR␣ KO mice. OVX decreased femoral, tibial, and ver-
LEE ET AL.
FIG. 5. Trabecular bone volume (BV/TV) of femur, tibia and vertebrae and cortical area of tibia measured by CT collected from WT and TCR␣ KO mice that were either SHAM or OVX. Values represent mean ± SE (n ⳱ 20–25/group). *Significant effect of OVX, p ⱕ 0.05.
FIG. 6. Static histomorphometric analysis of femurs from WT and nude mice that were either SHAM or OVX. Values represent mean ± SE (n ⳱ 9–12/group). *Significant effect of OVX, p ⱕ 0.05. +Significant difference between nude-SHAM and WTSHAM mice, p ⱕ 0.05.
tebral trabecular bone volume (BV/TV) by 35% in the femur, 35–37% in the tibia, and 37% in vertebrae compared with SHAM controls. OVX also decreased trabecular number and increased trabecular spacing in the femur, tibia, and vertebrae of both WT and TCR␣ ⌲⌷ mice by similar amounts (9–24% decrease in TbN, 13–36% increase in TbSp). CT analysis of cortical bone in the tibia found no difference in cortical bone area between WT SHAM and TCR␣ ⌲⌷ SHAM mice (Fig. 5). However, unlike nude mice, OVX significantly decreased tibial cortical bone area in both WT and TCR␣ ⌲⌷ mice by 5–11%. To further examine the role of T lymphocytes in OVXinduced bone loss, we performed static histomorphometry on the femurs from the same WT and nude mouse bones that were previously analyzed by DXA and CT (Fig. 6; Table 1). We found a significant decrease in cortical width in the femurs from nude-SHAM mice compared with WT SHAM mice. In addition, OVX induced cortical bone loss
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TABLE 1. STATIC HISTOMORPHOMETRY 4 WEEKS AFTER SHAM OR OVX TREATMENT
WT-SHAM WT-OVX Nude-SHAM Nude-OVX
OB S/BS (%)
OC S/BS (%)
N OC/BPm
TRACP+ area T/C
16.8 ± 1.7 19.4 ± 1.6 18.0 ± 1.4 15.4 ± 1.1
12.2 ± 1.3 13.8 ± 1.6 15.9 ± 1.5 15.9 ± 0.9
6.6 ± 0.4 7.2 ± 0.8 8.1 ± 0.4† 7.8 ± 0.7
1.00 ± 0.07 1.37 ± 0.15* 1.11 ± 0.13 1.74 ± 0.21*
Values are mean ± SE for 9–11 mice per group. * Significant effect of OVX, p < 0.05. † Significantly different from WT SHAM, p < 0.05.
in femurs from WT but not nude mice. Confirming the CT results, histomorphometry also found a 40% decrease in trabecular bone volume (BV/TV) in the femurs from nude SHAM mice compared with WT SHAM mice. Consistent with the CT results, OVX produced similar trabecular bone loss in WT (44%) and nude mice (48%). Significantly, we also found that OVX increased the relative TRACP+ area by 37% in WT and 57% in nude mice (Table 1) but did not alter other measurement of osteoblast or osteoclasts. Measurement of osteoclast surface per unit bone surface (OC S/BS) and the number of osteoclasts per unit bone perimeter (NOc/BPm) did not show an effect of OVX but did show that nude-SHAM femurs had increased NOc/ BPm compared with WT SHAM femurs. We also cultured bone marrow cells from WT and nude mice that were either SHAM or OVX and treated with or without M-CSF and RANKL (both at 30 ng/ml) for 5 days to stimulate osteoclast-like cell (OCL) formation (Fig. 7). In the SHAM groups, bone marrow cells from nude mice had significantly more (1.2-fold) OCLs in cultures than did similar cultures from WT SHAM mice (Fig. 7A). In addition, OVX increased OCL formation in bone marrow cultures by 1.3-fold in WT cells and 1.2-fold in nude cells compared with their respective SHAM controls. We further measured the number of colony forming unitgranulocyte macrophage (CFU-GM) in bone marrow of WT and nude mice that were either SHAM or OVX. This assay is an index of osteoclast precursor frequency(39) and was previously shown to be increased by OVX in mice.(43) As shown in Fig. 7B, nude SHAM mice had significantly more (1.4-fold) CFU-GM in their bone marrow cells than did WT SHAM mice. In addition, OVX significantly increased CFU-GM by 2.2-fold in WT and 1.8-fold in nude mice compared with their respective SHAM controls.
DISCUSSION We found that three different T lymphocyte–deficient mouse models, nude mice, RAG2 KO mice, and TCR␣ ⌲⌷ mice, lose trabecular bone mass after OVX at a rate that was similar to that of WT mice. However, the rate of loss of cortical bone mass in these animals was variable and dependent on the model and the bones that were examined. Because cortical bone mass accounts for ∼80% of the bone mass of the skeleton,(44) it is not unexpected that DXA, which measures only total bone mass, was unable to detect
FIG. 7. (A) Effects of OVX on OCL formation in bone marrow cultures from WT and nude mice that were stimulated with MCSF and RANKL (both at 30 ng/ml). Bone marrow cells were cultured for 5 days. (B) CFU-GM assay. Bone marrow cells from WT and nude mice that were either SHAM or OVX were cultured in semisolid methylcellulose to examine the number of osteoclast precursor cells. Values represent mean ± SE. *Significant effect of OVX, p ⱕ 0.05. +Significant difference between nudeSHAM and WT-SHAM mice, p ⱕ 0.05.
even large (>50%) changes in trabecular bone mass, when cortical bone mass was not also altered. This likely explains why we detected no change in total bone mass by DXA in femurs from nude mice after OVX because there was no significant change in cortical bone mass after 4 weeks as measured by CT (Fig. 4) or histomorphometry (Fig. 6). Trabecular bone is the most metabolically active(44) and is the most sensitive to estrogen withdrawal. Loss of trabecular bone after OVX probably resulted from an increase in osteoclast activity in both WT and nude mice because it was associated with similar increases in TRACP+ area in vivo and OCL formation and CFU-GM assay in vitro. Increases in TRACP+ area have previously been used to document the ability of OVX to increase total osteoclast
1710 activity.(30) Therefore, these results imply that T lymphocytes are not involved in the rapid increase in resorptive activity that is induced by estrogen withdrawal. Our results also show that reliance only on measurements of either total or cortical bone mass in these mouse models can lead to a confusing conclusion about the effects of OVX on bone mass. This is because assays of total or cortical bone mass seem to be less sensitive to changes after ovariectomy than are direct measurement of bone mass in the high turnover trabecular compartment.(44) Differences in the technique used to measure bone mass are likely responsible for the discrepancies among previous studies in nude mice, which used pQCT,(25) studies that examined changes in trabecular bone mass in nude rats by histomorphometry,(27) and our studies, which examined changes in bone mass by DXA, CT, and histomorphometry in multiple T lymphocyte–deficient mouse models. Like DXA, pQCT produces a relatively low resolution analysis of bone mass with a minimum voxel size of 100 m. In contrast, the CT that we used has a minimum voxel size of 12 m. It seems that only high-resolution imaging of trabecular bone either by CT or histomorphometry has adequate resolution to detect trabecular bone loss reliably after OVX in these mouse models. It was previously shown that nude mice have a delayed onset of puberty.(41) Hence, we wondered at the beginning of these experiments if this animal was a poor subject for studies of the effects of OVX on bone mass during its adolescence (roughly ages 5–10 weeks). However, our data showing that the uterine weight in nude SHAM mice was similar to that in WT SHAM mice and our findings that both WT and nude mice lost comparable amounts of uterine weight with OVX suggest that nude mice of this age are an appropriate model to examine the effects of Tlymphocyte depletion on OVX-induced bone loss. The results in TCR␣ KO and RAG2 KO mice confirm the nude mouse findings and show the variable ability of DXA or measurements of cortical bone mass by CT to assess bone loss after OVX in TLD mouse models. In contrast, measurements of trabecular bone mass by CT easily showed relatively large (35–37%) decreases in bone mass with OVX. TCR␣ KO mice maintain ␥␦ T lymphocytes, which may influence the response of bone mass to OVX. However, we doubt that ␥␦ T lymphocytes are involved in this response because RAG2 KO mice, lacking both ␣ and ␥␦ T lymphocytes, lost trabecular bone mass with OVX, as measured by CT at an equivalent rate to that of control C57BL/6 mice (Fig. 2). In addition, our previous study showed that OVX-induced vertebral and femoral bone loss in IL-7 KO mice, which lack both B and T lymphocytes, was identical to that seen in WT controls.(45) In contrast to a previous study,(25) we failed to show that OVX increased the percentage or number of T lymphocytes in the bone marrow of WT mice. The reason for this discrepancy is unknown but does not seem to be caused by strain, source, or age differences, because the strain (C57BL/6), source (Taconic), and age of the mice used in this study were identical to those used previously. In addi-
LEE ET AL. tion, a recent study confirmed our results and failed to show an increase in T lymphocytes in the bone marrow of mice after OVX.(46) The reasons for the differential effects of T lymphocyte depletion on estrogen-induced bone loss in the cortical and trabecular bone are also unknown but suggest that there are important effects of T lymphocytes on the cortical compartment. Possible reasons for these differences include effects of T lymphocytes on cortical remodeling or on osteoblast function as well as effects on osteoclasts. It is possible that the loss of T lymphocytes slows the rate of cortical bone loss with estrogen withdrawal and this is why we were able to more easily show loss of cortical and total bone mass in the ␣ TCD mice because these animals were examined 8 weeks after OVX rather than at 4 weeks after surgery as were the nude and RAG2 KO mice. Loss of T lymphocytes did seem to have some effects on bone mass independent of OVX because bone mass in nude and TCR␣ KO mice was decreased compared with their respective controls. This trend was significant for measurements of total BMD by DXA in both nude and TCR␣ KO mice and for histomorphometric measurements of cortical width and trabecular bone volume in nude mice. CT measurements in nude and TCR␣ KO mice did not show a significant decrease in bone mass in the SHAM groups except for values of trabecular bone in the femurs. Therefore, it seems that CT is a somewhat less sensitive measure of total bone mass in the basal state than is DXA. A possible explanation for this is that DXA is a more integrated measure of this parameter. In contrast, RAG2 KO mice, which lack both T and B cells, had increased vertebral bone mass and increased femoral cortical bone area. This finding suggests that loss of both T and B lymphocytes have effects on bone mass that differ from those seen in mouse models that lack just Tlymphocytes. Previously, it was found that in vitro(3) or in vivo(4) T-lymphocyte depletion was associated with increased in vitro osteoclast formation. These results suggest that T lymphocytes have an overall inhibitory effect on osteoclast formation, which is lost in nude and TCR␣ KO mice and results in lower bone mass in these animals. Consistent with this hypothesis, we found N Oc/BPm to be increased significantly in nude SHAM mice compared with WT SHAM mice, and in vitro OCL and CFU-GM formation was greater in nude SHAM bone marrow cells compared with WT SHAM cells. However, it is also possible that T lymphocytes regulate bone growth, and this response contributes to the lower total bone mass of some nude and TCR␣ KO bones. Our findings differ from the results of a previous study, which found that nude mice in a Swiss background had decreased osteoclast number (N Oc/BPm) in their bones with no significant difference in cortical or trabecular bone mass compared with WT mice.(47) This result together with the current findings suggests that there are strain-specific differences in the effects of the nude genotype on bone. Those authors also found increased bone-resorbing activity in the CM of splenic leukocytes from nude mice, which was associated with increased prostaglandin levels in the CM.(48) We also found that bone marrow cells from WT
T CELL–DEFICIENT MICE LOSE BONE MASS WITH OVARIECTOMY mice that were depleted of T lymphocytes produced increased OCLs in culture by a prostaglandin-mediated mechanism.(3) Together, these results imply that T lymphocytes regulate the production of factors, which influence bone resorption, and this mechanism may be involved in the differential bone-specific effects of T lymphocyte depletion on the responses of cortical and trabecular bone to OVX, which we identified in these studies. It is tempting to speculate that the recently identified population of memory T lymphocytes in bone marrow may be involved in this phenomenon.(49)
ACKNOWLEDGMENTS This work was supported by NIH/NIAMS Grant RO1AR4871401 from the U.S. Public Health Service. This work also used the CT core facility component of the Core Center for Musculoskeletal Research Grant NIH/NIAMS 5P30-AR046026 to the University of Connecticut Health Center.
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Address reprint requests to: Sun-Kyeong Lee, PhD Division of Endocrinology Department of Medicine MC-5456 University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030-5456, USA E-mail:
[email protected] Received in original form March 14, 2006; revised form July 18, 2006; accepted July 27, 2006.
