Age-related bone loss in mice is associated with an increased osteoclast progenitor pool

Bone, Vol . 15, No . 1, pp. 65-72, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA . All rights reserved 8756-3282/94 $6 .00 + .00

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Age-Related Bone Loss in Mice Is Associated with an Increased Osteoclast Progenitor Pool S . L . PERKINS,' R . GIBBONS, 2 S . KLING' and A . J . KAHN 2 ' Department of Pathology, University of Utah, Salt Lake City, UT 2 Department of Growth and Development, University of California San Francisco, San Francisco CA, USA Address for correspondence and reprints : Sherrie Perkins M .D ., Ph .D ., Department of Pathology, University of Utah School of Medicine, 50 N . Medical Drive, Salt Lake City, UT 84132, USA . Abstract

aging . This loss of bone may be especially debilitating in postmenopausal women, giving rise to osteoporosis and the resultant morbidity and mortality associated with increased incidence of fractures (Mosekilde 1992 ; Riggs & Melton 1986) . Maintenance of bone mass and density is dependent on the balance between two processes, bone matrix synthesis by osteoblasts and subsequent degradation by osteoclasts . Upon alteration in either or both of these two processes, bone density changes . Thus, bone loss could be due to lower osteoblastic numbers and/or activity, higher osteoclast numbers and/or activity or some combination of the above . Although the etiology of age-associated osteopenia is not well understood, it has been shown that osteoblastic function is decreased by the aging process in man (Parfitt 1987) and rodents (Bar-Shira-Maymon et al . 1989 ; Schapira et al . 1991 ; Silbermann et al . 1987) . However, the effects of senescence on osteoclast formation and function, as well as the role of the osteoclast in development of age-associated osteopenia, has not been established . The development of osteopenia may be due, in part, to increased osteoclast formation or osteoclastogenesis with age . Extensive experimental evidence suggests that osteoclasts are derived from a hematopoietic precursor cell that also ultimately gives rise to monocytes and macrophages (Burger et al . 1984 ; Kurihara et al . 1989 ; Marks & Popoff 1988 ; Udagawa et al . 1990) . These monocyte/macrophage/osteoclast precursor cells (MMOPCs) are responsive to a number of hematopoietic cytokines that are important in bone marrow cell proliferation and differentiation, including IL-3 (Kurihara et al . 1989 ; Lorenzo et al . 1987), GM-CSF (Barton & Mayer 1989 ; Kurihara et al . 1989 ; MacDonald et al . 1986) and M-CSF (Corboz et al . 1992 ; Hattersley et al . 1991 ; Kodama et al . 1991 ; Wiktor-Jedrzejczak et al . 1991) . MMOPCs at relatively early stages of maturation are sensitive to IL-3 and GM-CSF, whereas M-CSF acts at a relatively later stage and may be essential for terminal differentiation . One possible mechanism for increased osteoclast formation is an alteration in normal hematopoiesis that would lead to accumulation of MMOPCs in the bone marrow in aged individuals . Under appropriate conditions, these MMOPCs could differentiate to form osteoclasts . While age-associated changes in bone mass (Schapira et al . 1991) and bone formation have been extensively studied in the rat (Liang et al . 1992 ; Nishimoto et al . 1985), lack of purified cytokines and other techniques for study of the bone marrow prevents any conclusions about the bone marrow microenvironment's contribution to the development of osteopenia from being drawn . Similar age-associated bone loss

Age-associated osteopenia has been documented to occur in mice and, therefore, provides a model system whereby mechanisms of bone loss can be assessed in vivo and in vitro . One such mechanism, that could explain the increased resorptive activity seen in some forms of osteopenia, is an age-associated increase in the osteoclast precursor pool and osteoclastogenic formation . To test this hypothesis, we studied the bone marrow composition of aged (24 months) mice to determine if increased numbers of monocyte/macrophage/osteoclast precursor cells (MMOPC) were present when compared to young (4-6 months) animals . Our data show a moderate increase of 20-30% more hematopoietic cells obtained from the long bones of the aged animals . However, both liquid and semi-solid culture techniques demonstrate an approximately 2-3 .5-fold increase in the numbers of plastic adherent macrophages or mononuclear colonies in bone marrow derived from the aged mice when stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GMCSF) or macrophage colony stimulating factor (M-CSF), indicating a preferential increase in MMOPCs . In addition, cells derived from the aged mice show higher levels of c3ytokine stimulated incorporation of [ 3H]-thymidine and [ H]leucine, with increased protein synthesis seen up to 7 days after cytokine stimulation, suggesting that these cells also have an enhanced sensitivity to cytokines . Finally, coculture of bone marrow cells from aged mice with the ST-2 stromal cell line also gave rise to approximately twofold more tartrate-resistant acid phosphatase (TRAP) staining osteoclastlike multinucleated cells than did marrow from young animals which had the ability to degrade devitalized [ 3H]proline-labelled bone and produced cAMP following calcitonin stimulation . Collectively, these data suggest that in aged mice there is an increase in the numbers of MMOPCs that, under the proper conditions, give rise to increased numbers of osteoclasts . This may suggest a mechanism that could account for, in part, age-associated bone loss . Key Words : Osteoclast progenitors-Osteopenia-ST-2 coculture-Aging-M-CSF-GM-CSF-IL-3 . Introduction Bone loss and resultant skeletal structural weakening are welldescribed but poorly understood physiologic consequences of 65

66 has been seen in mice, where losses of both cortical and cancellous bone are seen (Bar-Shira-Maymon et al . 1989 ; Silbermann et al . 1987) . Using the aged mouse as a model system allows use of purified and recombinant murine cytokines, as well as wellestablished bone marrow culture techniques . To test the hypothesis that increased MMOPCs are present in aging animals, and may contribute to increased osteoclast formation and the development of age-associated osteopenia, we studied the bone marrow of both young and aged mice to determine if there were age-related alterations in the numbers of putative osteoclast precursor cells and osteoclastogenic potential . In addition, the responses of the MMOPCs to cytokines such as IL-3, GM-CSF and M-CSF, that have been implicated in osteoclast progenitor cell development, were determined using marrow cells from young and aged mice by colony formation in semisolid medium, cell proliferation rates and general protein synthesis . Finally, bone marrow from both young and aged animals was assessed for the capacity to form functional osteoclastlike cells using the ST-2 stromal coculture system (Udagawa et al . 1989), to determine if increased in vitro formation of osteoclast-like cells could be demonstrated in the bone marrow derived from aged mice . Materials and Methods Materials Recombinant murine growth factors including rGM-CSF, and rIL-3 were obtained from Genzyme (Boston, MA) . Murine M-CSF was purified from L929 serum-free conditioned medium by the method of Stanley (1985) . Whole fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY) . All other materials were obtained from Sigma (St . Louis, MO) unless otherwise indicated . Young (4-6 months) and aged (24 months) mice were purchased from the National Institute of Aging . Both the C57BL/6 and BALB/c strains were used in all experiments with essentially identical results . Histologic analysis The left femur from 6 each of young and aged mice were dissected and fixed in 10% buffered formalin overnight . Following decalcification in EDTA, the bones were processed and embedded in paraffin using standard techniques . Each bone was step sectioned with collection of every fifth section . Slides were stained with hematoxylin and eosin . All sections were examined using light microscopy and the maximal cortical bone thickness determined at the mid-diaphyseal shaft by micrometer on each section . The maximal cortical thickness observed for each animal was used to determine the mean maximal cortical thickness/ femur for each age group .

S . L . Perkins et al . : Aging mice have increased osteoclasts Ficoll-Hypaque density gradient centrifugation (d = 1 .07) . The light density cells were washed and plated as described for each experiment . For study of individual mice, the bone marrow was flushed from the left femur of the mouse and incubated for 4 h in growth medium in a 5% CO 2 atmosphere at 37° C to remove stromal cells . The nonadherent cells cell population was counted and 2 .0 X 105 cells/well from each animal were plated into 24-well plates (Nunc, Naperville, IL) in growth medium and incubated for the time indicated at 37° C in a 5% CO 2 humidified atmosphere . Cell counts (as described below) were performed on each of four replicate wells for each animal at the times indicated . Cell counts The nucleated cell population was determined by counting using a hemocytometer following dilution in a 4% acetic acid in water solution as well as by staining the cells with ethidium bromide/ acridine orange, which differentially stains viable cells, using an inverted fluorescent microscope . Cell counts were performed on the crude marrow preparation immediately after removal from the bone marrow cavity, following an overnight incubation to remove adherent cells and again subsequent to density gradient centrifugation . The adherent cell population was counted either following staining for a-napthyl butyrate esterase (nonspecific esterase) to determine the fraction of adherent, positively staining cells (typically >98% of all adherent cells) or by solubilizing washed adherent cells with 0 .0015% Zwittertergent and counting the nuclei in a hemocytometer or Coulter counter . For all experiments, at least three separate counts were made and averaged . [3H]-thymidine and [3H]-leucine incorporation Using a modification of the assay developed by Mochizuki et al . (1987), nonadherent cells were removed from primary bone marrow cultures that had been incubated overnight, counted and then resuspended at a concentration of 1 x 10 6 cells/ml in a-medium containing 15% FBS and the growth factor of interest . Aliquots of 0 .2 ml were plated into wells of 96-well microtiter plates (Costar, Cambridge, MA) and incubated at 37° C in a humidified 5% CO2 atmosphere . On days 3, 5 or 6 (as indicated), adherent mononuclear cells were pulse-labeled with either 1 µCi of [ 3H]methylthymidine (specific activity 6 .7 Ci/mmol) to determine proliferation rates or 1 µCi of [3 H]-leucine (specific activity 60 Ci/mmol) to determine general protein synthesis . All radioisotopes were purchased from New England Nuclear (Boston, MA) . Cultures were incubated with radioisotopes for 18 h . The cells were then harvested onto glass fiber filters with a Skatron cell harvester (Oslo, Norway) and incorporated tritium determined by liquid scintillation counting . Colony formation assay

Cell culture Primary cultures of bone marrow cells were done as previously described (Perkins & Teitelbaum 1991) . Briefly, pooled bone marrow cells were obtained from the femurs, tibiae and humeri by flushing the bone marrow cavity with cold a-modification of Eagle medium (a-medium) using the method of Tushinski et al . (1982) . Cell suspensions were incubated overnight at a density of 1 x 105 cells/ml in a-medium supplemented with 15% FBS and 500 units/ml M-CSF (growth medium) at 37° C in a 5% CO 2 humidified atmosphere . The nonadherent cell population was removed and the monocyte/macrophage precursor population enriched by collection of the light density cell fraction following

Soft agar colony proliferation assays were performed using a modification of the method of Bradley and Metcalf (1966) as previously described (Perkins & Yunis 1986) . Briefly, 2x a-medium supplemented with 40% fetal calf serum was mixed 1 :1 with a sterile 0 .6% agar solution (Difco, Detroit, MI) . Murine bone marrow cells were added to a final concentration of 5 X 104 (Ficoll-Hyaque mononuclear cell enriched bone marrow cells) or 1 x 10 (crude or unprocessed bone marrow cells) cells/ml . The cell suspension was mixed and 1 .0-m1 aliquots placed into 35-mm diameter tissue culture dishes (Corning, Corning, NY) containing 100 units of the relevant growth factor diluted to a final volume of 0 .1 ml in a-medium containing 20% FBS . The

S . L . Perkins et al . : Aging mice have increased osteoclasts plates were swirled to assure equal distribution of the cell suspension and growth factor . The dishes were incubated for 7 days in a humidified 5% CO 2 in air atmosphere at 37° C . Colony counts were performed using a dissecting microscope . Aggregates of 50 cells or more were scored as colonies . Following counting, the agar plates were fixed with 30% acetic acid in absolute methanol, followed by successive washes of 100%, 80% and 50% ethanol . The agar film was placed on a glass slide and air dried prior to staining with hematoxylin and eosin . Colony morphology was assessed using a microscope . In duplicate dishes, colonies were aspirated using a pipette, washed, placed on glass slides and stained for the presence of a-napthyl-esterase (nonspecific esterase) and napthol AS-D chloroacetate-specific esterase granules using standard histological techniques (Perkins & Teitelbaum 1991 ; Udagawa et al . 1989 ; Udagawa et al . 1990) .

Osteoclast-like cell generation Osteoclast-like cells were generated from bone marrow-derived precursor cells using the ST-2 coculture technique (Udagawa et al . 1989 ; Udagawa et al . 1990) . Early passage ST cells were plated at a density of 1 X 105/ml into 24-well plates (Nunc) and allowed to form an adherent monolayer for 24 h . Aliquots, 0 .1 ml, of light-density fraction bone marrow cells (at a density of 1 x 106 /ml), were then plated onto the stromal cell monolayer in the presence of 10 -8 M 1,25-dihydroxyvitamin D 3 (a gift from Dr . M . Uskokovic of Hoffman-LaRoche) and 10 - ' M dexamethasone . Fresh media and hormones were added every fourth day . On day 12, the cells were fixed with methanol : acetone (1 :6 volume:volume) and then washed with distilled water and air dried . The cells were then stained for tartrate-resistant acid phosphatase activity (TRAP) using 0 .1 M sodium acetate containing tartrate, napthol AS-BI phosphoric acid in N, N-dimethyl formamide and 4% pararosaniline for 30 min, then washed with distilled water and allowed to air dry (Janckila 1978) . The multinucleated TRAP-positive osteoclast-like cells were scored using an inverted microscope . To measure the functional characteristics of the osteoclastlike cells formed, bone marrow cells from young or aged animals were cocultured with ST-2 stromal cells as outlined above . To determine the bone resorbing capacity of the osteoclast-like cells degradation of bone collagen was measured by the method of Blair et al . (1986) . On day 12 of incubation, 100 µg (20 pCi) of ['H]-proline-labelled devitalized rat bone, prepared by the method of Teitelbaum et al . (1979), was added to each well containing osteoclast-like cells following coculture of ST-2 cells and murine bone marrow cells . All conditions tested had four to eight identical wells tested . Following incubation for 3 days, the supernatants were removed, filtered to remove any bone particles and the amount of [ 3H]-labelled peptides released by osteoclastic degeneration of the bone particles determined following scintillation counting . An aliquot of bone was also added to control wells containing no cells, ST-2 cells alone and bone marrow cells (which were >99% nonspecific esterase-positive macrophages) to determine background release of ['H] . In all experiments the nonspecific release from the medium alone was less than 8% of the total counts (range 5-8%) . Minimal release was noted from the wells containing the ST-2 cells (<0 .5% of total counts, range 0 .16-0 .43%) . Additional controls of untreated bone marrow macrophages alone as well as those treated with the same levels of vitamin D and dexamethasone on the same schedule as the ST-2 coculture wells also showed minimal ['H]proline release (<0 .2% of the total counts, range 0 .0-0 .2%) after correction for nonspecific release . A final control, in sep-

67 arate experiments, showed that addition of 10-20 nM calcitonin (Sigma) inhibited 85-95% of all labelled proline release in both young and aged animals . All controls and wells were performed in quadruplicate, and the maximal value observed from these controls was subtracted from values obtained in the wells containing osteoclast-like cells to reflect bone degradation rather than nonspecific leaching out of counts or nonspecific cellular degradation of bone particles . In all cases, the counts observed in control wells were less than 8% of the counts observed in the wells containing osteoclast-like cells . In addition, similarly prepared osteoclast-like cells were tested for calcitonin-dependent cAMP production by the method of Takahashi et al . (1988) . Briefly, the media was removed and fresh a-MEM containing 0 .1% bovine serum albumin and 1 mM isobutylmethylxanthine added, then incubated at 37° C for 10 min . Salmon thyrocalcitonin, 10 nM, was added to the appropriate wells and incubated for 15 min at 37° C . The supernatants were removed and the cell layers extracted with 95% ethanol containing 1 mM HCI . The cell layers were dried in a boiling water bath and resuspended and the cAMP levels determined using a radioimmunoassay (Amersham, Rockford, IL) . Background levels were determined from cells, which were not treated with calcitonin and subtracted from the stimulated cells, but represented < 1 % of the counts observed following calcitonin stimulation .

Results To confirm that aged mice have age-associated loss of bone mass, femurs were isolated from aged (24 months) and young (6 months) C57BL/6 mice and examined following formalin fixation, EDTA decalcification and routine histologic processing . As seen in Figure 1, the femurs of aged mice show a marked diminution in cortical as well as trabecular bone . Measurements of the cortical bone found an average thickness of 1 .2 ± 0 .16 mm in the mid-diaphyseal shaft in the aged mice as compared to 1 .4 ± 0 .12 mm in the young mice (p < 0 .05, n = 6 of each age group) . In addition, the aged mice had decreased trabecular bone (both in number of trabeculae as well as in trabecular mass), and their trabeculae were attenuated when compared to the younger mice (see Fig . 1A and B [young mice] compared to Fig . 1C and D [aged mice]) . Our initial studies examined the bone marrow that could be isolated from the long bones of animals from both age groups . The bone marrow was studied from both individual (Table I) and pooled (Table II) animals, with essentially similar results . As seen in Table I, each of 5 individual aged (24-month-old) mice contained increased numbers of bone marrow cells/femur when compared to the control young (3-month-old) mice, similar to that seen in experiments where the bone marrow from 3 or more animals was pooled (Table II) . In all experiments, there was an approximately 20-40% increase in the amount of bone marrow that was isolated from aged animals (p < 0 .01), perhaps reflecting the expanded medullary hematopoietic compartment due to bone loss . When bone marrow cellularity was normalized for animal body weight, the age-associated increase in cellularity was not significant in the pooled bone marrow experiments . When identical numbers of bone marrow cells from the young and aged animals were plated in the presence of M-CSF to stimulate the growth and differentiation of the MMOPCs, the aged mice showed a two- to three-fold increase in the numbers of plastic adherent, nonspecific esterase-positive macrophages at days 2 and 3 of incubation (p < 0 .005) (see Tables I and II) . In addition, the percentage of bone marrow cells that gave rise to macrophages was increased in the aged mouse by about 2 .0- to

68

S . L . Perkins et al . : Aging mice have increased osteoclasts

A.

C.

Fig. 1 . Aged mice develop osteopenia . Femurs from C57BL/6 male mice were dissected, fixed, sectioned and stained with hematoxylin and eosin . Photomicrographs of young (6 months) femurs are shown in (A) (40X) and (B) (100X), while those from the aged (24 months) mice are depicted in (C) (40X) and (D) (100x) .

2 .5-fold (p < 0 .005) when compared to the younger controls (see Tables I and II) . The similar results observed using both individual and pooled mouse marrows suggest that the increase in MMOPCs is related to age-associated changes in bone marrow composition, rather than contribution by a single aberrant animal . Based on these findings, pooled bone marrow was used for subsequent experiments to allow sufficient amounts of marrow to be studied . To further confirm that the aged mice had increased myelomonocytic activity, soft agar colony formation assays were performed using several cytokines known to act in proliferation and differentiation of MMOPCs (rIL-3, rGM-CSF and M-CSF) . As seen in Figure 2, there was a marked increase in bone marrow colony forming capacity in the aged mice when compared to the young controls, despite identical numbers of cells being plated . rIL-3 had the most marked effect with a 2 .4-fold increase in colonies using the bone marrow from aged mice (p < 0 .001), followed by rGM-CSF with a twofold increase (p < 0 .05) and M-CSF with a 1 .8-fold increase (p < 0 .05) .

Correlated with the increase in colony formation seen above, the bone marrow precursor cells derived from aged mice showed a persistence of proliferation, as measured by [ 3H] thymidine incorporation, following stimulation with 100 units/ml of rGMCSF (see Fig . 3A) . Initially, bone marrow cells derived from young mice showed an equally as high level of [ 3H]-thymidine incorporation at 3 days, but this level decreased by day 7 of culture . In contrast, the bone marrow cells derived from aged mice showed persistently high levels of [ 3H]-thymidine incorporation at day 5, which did not return to young control levels until day 7 of culture (p < 0 .05 at day 3 and (p < 0 .007 at day 5) . In addition, bone marrow cells derived from the aged mice showed high levels of protein synthesis, as measured by [3 H]leucine incorporation, which remained elevated two- to four-fold (p < 0 .005 at day 5 and day 7) over those measured in the bone marrow cells of the young mice throughout the course of the experiment (see Fig . 3B) . These increased levels of DNA and protein synthesis suggest that the cells derived from the aged mice remain metabolically stimulated following cytokine expo-



S . L . Perkins et al . : Aging mice have increased osteoclasts

69

Table I . Bone marrow MMOPCs are increased in individual aged mice

Wt . (g)

Crude marrow Cells/femur (x 10 6 )

Cells/femur/g body wt . (x 10 6)

Plastic adherent esterase positive progeny Day 2 Day 3 Cells/well % Total cells Cells/well % Total cells (x 104 ) plated (x 104) plated

Young #1 #2 #3 #4 #5 Young mean

26 .3 24 .2 28 .2 27 .7 24 .5 26 .2 ± 1 .8

23 .4 23 .5 22 .8 27 .4 21 .6 23 .7 ± 2 .0

0 .89 0 .97 0 .81 0 .99 0 .88 0 .91 ± 0 .7

2 .8 4 .1 2 .5 2 .4 2 .3 2 .8

± ± ± ± ± ±

0 .8 0 .4 0 .7 0 .7 0 .3 0 .7

9 .3 13 .7 8 .3 8 .0 7 .6 9 .4 ± 2 .2

4 .6 5 .2 6 .7 4 .7 6 .3 5 .5

± ± ± ± ± ±

0 .4 0 .7 0 .5 0 .6 0 .9

15 .3 17 .3 22 .3 15 .6 21 .0 18 .3-±2 .8

Aged #1 #2 #3 #4 #5 Aged mean

32 .4 30 .8 32 .1 30 .7 30 .7 31 .3 ± 0 .8b

33 .0 34 .8 34 .3 29 .4 36 .8 33 .7 ± 2 .4 a

1 .02 1 .13 1 .07 0 .96 1 .20 1 .08 ± 0 .9 1

6 .7 6 .9 6 .4 7 .1 6 .7 6 .8

± ± ± ± ± ±

0 .7 0 .7 0 .6 0 .7 0 .8 0 .38

22.3 23 .0 21 .3 23 .6 22 .3 22 .5 ± 0 .8a

11 .6 10 .0 12 .6 11 .9 11 .6 11 .5

± ± ± ± ± ±

0 .7 0 .6 0 .9 1 .4 0 .6 1 .0 1

38 .7 33 .3 42 .0 39 .7 38 .7 38 .5 ± 2 .91

0 .5

Bone marrow cells were obtained from the left femur of 5 young (6 months) and 5 aged (24 months) C57BL/6 mice . The number of cells obtained from each femur was determined by hemocytometer . The marrow from each mouse was plated individually at a density of 2 X 10 5 cells/well in the presence of 500 units/ml M-CSF . The number of plastic adherent, nonspecific esterase-positive cells was determined on day 2 and 3 after plating . The number of cells/well is the mean of four identical wells from each mouse's bonemarrow ± standard deviation . The mean of all the aged and all the young mice is also shown, ± standard deviations . Statistical significance of differences between young and aged animals is shown (ap < 0 .001 ; by < 0 .005) . sure longer than their younger counterparts, suggesting that these cells have altered cytokine responsivity when compared to young controls . To test the hypothesis that the increased levels of MMOPCs present in the aged mouse is paralleled by an increase in osteoclastic-forming potential under the proper cytokine conditions, we made use of the ST-2 stromal coculture technique . When identical numbers of bone marrow cells were plated in this osteoclast-stimulating microenvironment, the cultures derived from the marrow of aged mice gave rise to 2 .5-fold more TRAPpositive osteoclast-like cells than did the cultures from young mice (see Fig . 4, (p < 0 .001) . No significant difference in osteoclast-like cell size, degree of multinucleation or other morphologic parameters was noted between the cells derived from the young or aged animals . To determine if tie TRAP-positive cells being formed by the

ST-2 coculture system had the ability to function as osteoclastlike cells, two separate functional assays were performed . First, the osteoclast-like cells were tested for the ability to degrade [3H]-proline-labelled devitalized rat bone . As seen in Figure 5, the osteoclast-like cells formed from both young and aged animals had the capacity to degrade the radiolabelled bone with subsequent release of the [ 3H]-labelled peptides derived from bone matrix into the medium . As expected by the higher levels of osteoclast-like TRAP-positive cells observed in earlier cultures, the cultures derived from the aged mice degraded more bone (24 .2 .g/well p vs . 18 .5 .g/well p ; p < 0 .004) . In contrast, control cultures containing ST-2 cells alone degraded 0 .04 µg/ well, and those with bone marrow macrophage cells degraded 0 .01 .g/well . The overall 25% increase in bone resorption seen in this experiment is less than might be predicted from the twoto four-fold increase in TRAP-positive cells observed . In addi-

Table II . Bone marrow MMOPCs are increased in the pooled bone marrow of aged mice

Expt . # 1 Young Aged Expt . #2 Young Aged Expt . #3 Young Aged

Crude marrow At Isolation Cells/mouse Cells/g body weight X 10 6 X 106

Plastic adherent esterase-positive progeny Day 2 Day 3 % Total Cells/mouse % Total marrow cells x 106 marrow cells

Cells/mouse X 106

57 .3 ± 1 .3 71 .1 ± 2 .l b

2 .19 ± 0 .02 2 .37 ± 0 .04c

3 .7 ± 0 .4 13 .0 ± 0 .6'

6 .4% 18 .4%

6 .5 ± 0 .7 19 .9 ± 1 .2a

11 .3% 27 .9%

39 .7 ± 0 .6 54 .1 ± 1 .86

1 .56 ± 0 .06 1 .86 ± 0 .04`

2 .9 ± 0 .5 8 .8 ± 0 .7b

7 .3% 16 .3%

6 .1 ± 0 .4 21 .2 ± 1 .61

15 .4% 39 .2%

64 .5 ± 1 .9 79 .6 ± 2 .7b

2 .48 ± 0 .08 2 .74 ± 0 .07`

4 .0 ± 0 .9 13 .9 ± 0 .71

6 .2% 17 .5%

10 .3 ± 0 .7 23 .6 ± 1 .8b

12 .9% 29 .7%

Bone marrow cells were obtained from the femurs, tibias and humeri of young (6 months) and aged (24 months) mice . The number of nucleated bone marrow cells isolated/mouse was determined by hemocytometer . Identical numbers (2 X 105 cells/well) of cells were plated in the presence of 500 units/ml of M-CSF . The number of plastic adherent, nonspecific esterase-positive cells was determined on day 2 and 3 after plating . All results are normalized to a single mouse ± standard deviation . In experiment #17 mice, in experiment #2 3 mice, and in experiment #3 5 mice from each age group were pooled and studied . Statistical significance of differences between young and aged animals is shown (p < 0 .001 ; by < 0.005 ; `p < 0 .01) .



S . L . Perkins et al . : Aging mice have increased osteoclasts

70 200

1

O Young ∎ Old 00

0

U

collectively, aged animals showed a moderate increase in the total number of bone marrow cells present in long bones-a finding in keeping with the enlarged bone marrow cavity area associated with loss of trabecular and cortical bone . Moreover, in vitro growth of bone marrow cells derived from aged animals showed a significant, disproportionate increase in the numbers of MMOPCs when compared to younger controls . This increase was seen both in cytokine-stimulated liquid cultures that gave rise to adherent macrophages as well as by increased mononuclear colony formation in semisolid cultures . The enhanced mononuclear colony forming activity was evident in the presence of IL-3, GM-CSF and M-CSF, growth factors that impact on

0 Control IL-3 GM-CSF M-CSF Fig. 2. Mononuclear colony formation is enhanced in the bone marrow derived from aged mice . Semisolid agar colony forming assays were performed by plating 1 x 10 5 bone marrow cells/ml from either young (4 months) or aged (24 months) animals in the presence of 100 units of the indicated cytokine . Colony counts were performed at day 6 of incubation using an inverted microscope . A colony was defined as a distinct aggregate of >50 cells . All cultures were performed in triplicate, and the values plotted represent the mean ± standard deviation . The data shown is a representative experiment of three performed . Significance was determined using the Student's paired t test (*p < 0 .001, **p < 0 .05) .

A

tion, the TRAP-positive osteoclast-like cells were also tested for calcitonin-dependent cAMP production . As seen in Figure 6, there was a slight increase in the levels of cAMP production by the cultures of osteoclast-like cells derived from the aged mice when compared to the young mice (p < 0 .025) . However, the increase in cAMP levels was, again, lower than would be predicted by the numbers of TRAP-positive cells observed . 3

5 Days in Culture

7

3

5 Days in Culture

7

Discussion

B Previous work has shown that mice are susceptible to ageassociated loss of bone mass and may, therefore, provide a model system for study of this process as it occurs in other mammals as well as man (Bar-Shira-Maymon et al . 1989 ; Silbermann et al . 1987) . Histomorphometric studies have shown loss of both cortical and trabecular bone in the lumbar vertebrae (Bar-Shira-Mayon et al . 1989) and femoral cortical bone (Silbermann et al . 1987) . The aged mice in this study also showed similar losses in femoral cortical bone by gross examination and limited histologic analysis . Bone loss requires an excess of osteoclast-mediated bone resorption compared to new bone formation by osteoblasts . Work by the Bar-Shira-Maymon (1989) and Silbermann (1987) groups have shown that osteoblastic number and function is decreased in the aged mouse, similar to findings in man (Parfitt 1987) . We hypothesized that, in addition to the decrease in osteoblastic number and function, increased osteoclastic activity may also be an essential component in the development of osteopenia . As osteoclasts are derived from a hematopoietic precursor cell that also gives rise to monocytes and macrophages (Burger et al . 1984 ; Kurihara et al . 1990; Marks & Popoff 1988 ; Udagawa et al . 1990), we studied the pool of available MMOPCs in aged animals using liquid and semisolid agar proliferation assays to determine the numbers of cells present which were responsive to cytokines that have been implicated in osteoclastic differentiation such as IL-3, GM-CSF and M-CSF (Barton & Mayer 1989 ; Corboz et al . 1992 ; Hattersley et al . 1991 ; Kodama et al . 1991 ; Kurihara et al . 1989 ; Lorenzo et al . 1987 ; MacDonald et al . 1986 ; Wiktor-Jedrzejczak et al, 1991) . As documented above, when studied both individually and

Fig . 3 . Increased [ 3 11-thymidine and [ 3 H1-Leucine incorporation is seen in the bone marrow derived from aged mice . Bone marrow cells derived from either young (4 months) or aged (24 months) mice were plated into 96-well plates at a density of 2 x 10 5 cells/ml (0 .2 ml) in the presence of 100 units/well of GM-CSF . On the day indicated, the cultures were pulsed for 24 h with I µCi/ml of either [ 3 H1-thymidine (A) or [ 3 H1leucine (B) . The number of cell-associated counts was determined by scintillation counting . The data are the mean of triplicate cultures ± standard error. Significance was determined using the Student's paired t test .



S . L . Perkins et al . : Aging mice have increased osteoclasts

U 500

71

* p<0 .001

2 N O Q LL Q

Young Old Fig . 4 . Increased numbers of osteoclast-like cells are formed from the bone marrow of aged mice . Osteoclast-like cells were generated in vitro by use of the ST-2 stromal coculture technique . From either young (4 months) or aged (24 months) animals I X 105 bone marrow cells/ml were seeded onto a monolayer of ST-2 cells in 24-well plates . The cultures were incubated for 14 days in the presence of 10-s M 1,25(OH)2 vitamin D3 and l0-7 M dexamethasone with changes of the media every fourth day . Cultures were stained for TRAP activity and the number of TRAPpositive multinucleated cells (MNC) determined by counting with an inverted microscope . Values shown are the mean of four identical wells ± standard deviation . Data was analyzed using the Student's paired t test (*p < 0 .001) . The experiment shown is representative of four performed. various stages of the hematopoietic cascade leading to the differentiation of monocytes, macrophages and osteoclasts . The age-associated increase in IL-3 stimulated colony formation was at slightly higher levels than was seen with either GM-CSF or

Young Old Fig. 5 . Bone degradation by osteoclast-like cells . Osteoclast-like cells from the bone marrow of young and aged mice were generated in vitro by the ST-2 coculture technique . At day 14, 100 mg of [3H]-prolinelabelled bone was added to each well . Following 3 days of incubation, the supernatants were collected, filtered and the amount of [3H]-labelled bone matrix-derived peptides released into the medium determined by scintillation counting . Counts were corrected for nonspecific release of label by subtraction values obtained from wells containing medium alone . The values shown are the mean of four identical wells ± standard deviations (the error bars are too small to be detected in the bar representing the aged mice) . Data was analyzed using the Student's paired t test (*p < 0 .004) . The experiment shown is representative of three performed .

Young Old . 6 Fig . Calcitonin-dependent cAMP production by osteoclast-like cells . Osteoclast-like cells from the bone marrow of young and aged animals were formed using the ST-2 coculture technique . At day 14 of incubation, the cells were stimulated with 10 nM salmon calcitonin and the cell-associated cAMP extracted and assayed by radioimmunoassay . The values shown are the mean of four identical wells ± standard deviation . Data was analyzed using the Student's paired t test (*p < 0 .025) . The experiment shown is representative of three performed . M-CSF, which may suggest increased sensitivity to this cytokine by the marrow cells derived from the aged mice . This heightened sensitivity may be important in maintenance of a pool of early precursor cells which could give rise to multiple progeny types . Further characterization of the bone marrow MMOPCs derived from the aged mice found both prolonged stimulation of [3H]-thymidine and [3H]-leucine incorporation following cytokine stimulation with GM-CSF . This finding suggests that the increase in colony formation noted above may be due, at least in part, to enhanced cytokine responsivity by the precursor cells, allowing for increased clonal expansion . Osteoclast generation assays done using the ST-2 stromal cell line suggest that the apparent increases in MMOPCs seen in the bone marrow of aged mice can give rise to increased numbers of TRAP-positive osteoclast-like cells in the proper microenvironment . The increased level of osteoclast-like cell formation seen in this system mirrors the increased colony formation data (approximately 2-2 .5-fold increases) seen in other assays . In addition, the osteoclast-like cells generated from the aged mice showed increased levels of bone resorption as measured by [3H]proline-labelled bone degradation as well as increased levels of calcitonin-dependent cAMP production . Both of these functional assays showed levels of osteoclastic activity lower than would be predicted by the numbers of TRAP-positive cells formed . This may indicate functional differences in osteoclast-like cells derived from the aged animals or may reflect increased numbers of TRAP-positive cells which do not function as osteoclasts being formed in cultures containing the cells from the aged animals . It has been noted that TRAP positivity does not always correlate with osteoclasts, and that some nonosteoclastic cells such as fused peritoneal macrophages may also stain positively (Hattersly & Chambers 1989) . Further studies are in progress to define the functional characteristics of the osteoclast-like cells formed from both young and aged animals and to better define the phenotype of the TRAP-positive cells formed by both the aged and young bone marrow derived cells .

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The data presented above suggest that aging mice have an increased pool of progenitor cells that can give rise to monocytes, macrophages or osteoclast-like cells . The ultimate lineage fate of these cells would then be determined by local levels of cytokines and other factors . In a microenvironment conducive to the formation of osteoclast-like cells, such as the ST-2 stromal coculture system provides, increased levels of osteoclast-like cells were formed by cells isolated from the aging mouse . The increased MMOPC progenitor pool provides an apparent mechanism whereby the aged animals may form more osteoclasts under the proper conditions . This, coupled with observed decreases in osteoblastic function may be, in part, instrumental in the development of age-associated osteopenia .

Acknowledgment : We wish to thank Debbie Meichle for performing the TRAP, nonspecific esterase and specific esterase stains . This work was supported by the National Osteoporosis Foundation (S . L . P .) and NIH RO1-33716 (A . J . K .) . A portion of this work was presented at the American Society for Bone and Mineral Research meetings in Minneapolis, MN in October 1992 .

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Date Received : January 5, 1993 Date Revised : July 19, 1993 Date Accepted : August 9, 1993