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ELSEVIER SCIENTIFIC PUBLISHERS IRELAND
Bone and Mineral 23 (1993) 317-332
Immune cell defects affect bone remodelling in osteopetrotic op/op mice C. Philippart a, E. Tzehoval b, Y. Moricard a, A.-F. Bringuier a, C. Seebold a, F.-M. Lemoine c, A. Arys d, N. Dourov d, M.-L. Labat *a aCNRS, Laboratoire de Physiopathologie Osseuse, Centre de Recherches Biom~dicales des Cordeliers, 15 Rue de l'Ecole de Medecine, 75270 Paris Cedex 06, France hDepartment of Cell Biology, The Weismann Institute of Science, Rehovot, Israel CDEpartement d'hEmatologie, HSpital St-Antoine, 75012 Paris, France dLaboratoire d'Anatomie Pathologique et de Microscopie Electronique, Facult~ de MEdecine, Universit~ Libre de Bruxelles, Campus Erasme B6t. C, 808 route de Lennick, B-1070 Brussels, Belgium
(Received 4 May 1993; revision received 5 August 1993; accepted 1 September 1993)
Abstract The aim of the present work was to further characterize immunological defects in osteopetrosis. The op/op mutant mouse is of particular interest since a marrow cavity develops spontaneously in older animals. The interleukin production (IL-I, IL-2, IL-3, IL-4, IL-6), the presence of macrophage differentiation antigens and the evolution of the bone lesions were studied in osteopetrotic and normal mice. Low levels of IL-1, IL-3 and IL-4 were observed at the age of 6 weeks in the op/op mice. However, at 22 weeks of age, the level of IL-1 and IL-4 returned to normal value in these op/op mice whereas the level of IL-3 remained partially decreased at the same age. Furthermore, macrophage expression of MAC-2 antigen, reduced at 12 weeks of age was found to be normal 10 weeks later. These immunological defects and their recovery seems to be concomitant with the healing of the bone lesions. Key words: Osteopetrosis; Interleukins; Macrophages
1. Introduction Osteopetrosis is characterized by a uniformly dense appearance of most of all long bones at X-ray examination [1]. This is due to a failure in development o f the mar* Corresponding author. 0169-6009/93/$06.00 9 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0169-6009(93)00636-H
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row cavity as a result of deficient osteoclastic bone resorption [2]. Two main causes of deficient bone resorption are recognized; either the defect lies in hematopoietic stem cells with osteolytic capabilities or in the microenvironment, unable to provide appropriate signals for mononuclear cells proliferation and/or differentiation into osteoclasts [3-8]. Following the demonstration by Walker in 1973 [3] of the hematopoietic origin of the disease, further studies by Labat and coworkers led to reconsideration of osteopetrosis as an immune disease [9,10]. Osteopetrosis, either inherited as in the op/op mutant rat or experimentally induced by a biphosphonate treatment, was associated with an early atrophy of the thymus and an impairment of T-cell functions [11]. A single bone marrow injection cured the immune deficiency and the bone lesions of the osteopetrotic rat [5]. However, op/op mice could not be cured by such treatment, suggesting that the defect lies in the bone marrow environment [7]. Indeed, it was shown that cultured osteoblasts and fibroblasts from op/op mice did not produce and secrete macrophage colony-stimulating factor (M-CSF) [12,13]. MCSF is necessary for the growth and differentiation of mononuclear phagocytes [14] which are considered to be osteoclast precursors [15]. It was suggested that M-CSF cooperates with other cytokines, such as IL-3 and IL-1, in the formation of osteoclasts [16]. Thus, the op/op mouse could serve as an excellent model to study the biology of interleukins in relation to bone resorption and to macrophage differentiation. In this study, we report that besides defective production of M-CSF documented by Wiktor-Jedrzejczak et al. [12] and Felix et al. [13], interleukin secretions are impaired in op/op mice. Low levels of IL-1, IL-3 and IL-4 are observed at the age of 6 weeks. Between 12 and 22 weeks, IL-1 and IL-4 reach almost normal levels, whereas IL-3 remains partially decreased at 22 weeks of age. Furthermore, in older mutants, we observe normalisation in the op macrophage ultrastructure and phenotype. On the other hand, the development of long bone marrow cavities is also noted. We can not assert that the increased cytokine levels cause increased osteoclast activity. Nevertheless, normal levels of cytokines and changes in ultrastucture and phenotype of macrophage are concomitant with the healing of bone lesions. These results suggest that increased levels of IL-1, IL-3 and IL-4 may not only reflect a change in macrophage-modulating activity but that these cytokines may also be involved in bone remodelling. 2. Materials and methods 2.1. Mice
Osteopetrotic (op/+) heterozygotes-tested breeding pairs were purchased from the Jackson Laboratory (Bar Harbour, ME). Animals were maintained in controled environment modules (ESI-Flufrance Cachan, France). Mating heterozygotes results in the production of op/op and normal littermates (+/+ or +/op). The op/op mutant mice are recognized by the absence of incisor teeth as soon as 7 days after birth [17]. After weaning, animals were fed on a standard sterile diet RO3-40 (UAR Villemoisson Orge, France) given as a powder to the mutants. Four mutant mice (op/op) and 4 controls (including +/+ and +/op genotypes) were individually tested in each age group: 6, 12 and 22 weeks.
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2.2. Reagents The following culture media or reagents were used throughout the experiments. Thioglycollate broth was prepared according to the manufacturer's instructions (Difco, Detroit, MI). Culture media such as PBS Dulbecco's, RPMI 1640, DMEM and fetal calf serum were purchased from Gibco (Grand Island, NY). Heparin: thromboliquine was from Organon Teknika BV (Boxtel, The Netherlands). Mitogens such as lipopolysaccharide (LPS) from E. coli 0111 :B4 (Sigma, St-Louis, MO), concanavalin A (ConA) (ICN Immunobiologicals, Lisle, IL) and phytohemagglutinin (PHA) (WeUcome, Dartford, UK) were used. Cells were pulsed with tritiated thymidine, specific activity 23 Ci/mmol (Dositek, Orsay, France). All cell cultures were performed in 24- or 96-well microplates (Costar, Cambridge, MA). Specificity for each bioassay was tested using recombinant interleukins and antiinterleukins (Genzyme, Boston, MA). Cell lines used in the bioassays were from ATCC for LBRM-33-1A5 and CTLL.B9 and CT.4S were a generous gift from W. Paul (NIH) and MC/9 was kindly provided by the Weizmann Institute of Science. 2.3. Thioglycollate induced peritoneal macrophages (TIP macrophages) Mice were injected intraperitoneally (i.p.) with 3 ml of thioglycollate broth. Four days later, the mice were killed and injected i.p. with 10 ml of PBS Dulbecco's containing 5 units heparin/ml. The peritoneum was gently massaged and the peritoneal fluid was aspirated with a 10 ml syringe. The cell suspensions were centrifuged at 4~ at 200 x g for 10 min. The supernatant was discarded and the cells were resuspended in PBS. Cell preparation consistently contained > 90% macrophages, of which more than 90% were viable cells (Trepan blue die exclusion). 2.4. Spleen cells Spleens, once aseptically removed from op/op or normal littermates at different ages, were flushed by injecting PBS into the organ. The recovered cells were then seeded in 24-well microplates at the concentration of 10 7 cells/well. Production of IL-1 and IL-6 was determined following overnight (18 h) culture in DMEM supplemented with 40#g/ml gentamycin in the presence of 30#g/ml LPS. Production of IL-2, IL-3 and IL-4 was determined following overnight (18 h) culture in RPMI 1640 supplemented with 10% FCS, 5 x 10-5 M 2-mercaptoethanol (2-ME) and 40#g/ml gentamycin in presence of 10#g/ml ConA. The supernatants were centrifuged at 900 x g for 10 min, then serially diluted and assayed in triplicates for interleukins. 2.5. Bioassays of interleukins Supernatants prepared from in vitro cultures of mitogen-activated spleen cells were bioassayed for the following interleukins: IL-1, IL-2, IL-3, IL-4 and IL-6, using selected responsive cell lines. Experiments were all performed in 96-well microplates. Each well contained a constant amount of diluted supernatant (100#1) and I00#1 of cells. Recombinant cytokines were used as positive controls for bioassays. For each bioassay, the specificity was tested using antibodies directed against the cytokine study. The cytokine linked to its specific antibody is unable to stimulate cell proliferation. IL-1. A thymoma cell line LBRM-33-1A5, was used to assay for IL-1, based on the ability of IL-1 to augment IL-2 production as a costimulant with PHA [18].
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Several triplicated dilutions of supernatants from LPS-activated spleen cells were added to the culture medium of 5 x 10 4 cells/well LBRM-33-1A5 in presence of PHA at a final concentration of 3#l/ml. After 18 h, IL-2 activity was determined by removing 100/~l samples from the wells and incubating them with 100 ttl of IL-2dependent continuous T lymphocytes line, CTLL (5 x 10 3 cells/well) [19] for an additional 20 h, followed by a 12-h pulse with 1/~Ci/well of tritiated thymidine and harvested. An IL-2 standard mouse factor, prepared from ConA-stimulated mouse spleen cells, was used as a positive control. Results were expressed in counts/rain. IL-2. Proliferation of the IL-2-dependent CTLL indicator cells was determined as described previously [19]. Briefly, triplicated dilutions of supernatants from ConAactivated spleen cells were added to the culture medium of 5 x 10 3 cells/well CTLL indicator cells, in RPMI supplemented with 10% FCS and 5 • 10-5 M 2-ME. After 20 h the cultures were pulsed (12 h) with 1/~Ci/well of tritiated thymidine and harvested. IL-3. Several triplicated dilutions of supernatants from ConA-activated spleen cells were added to the culture medium of 5 • 10 3 cells/well MC/9 indicator cells [20], in DMEM supplemented with 10% FCS and 5 • 10-5 M 2-ME. After 48 h the cultures were pulsed (18 h) with 1 t~Ci/well of tritiated thymidine and harvested. IL-4. The highly responsive CT.4S cell line was used to assay IL-4 activity in ConA-activated spleen cell cultures [21]. Several triplicated dilutions of the supernatant in RPMI supplemented with 10% FCS and 5 x 10-5 M 2-ME and 0.4 mg/ml gentamycine, were used as culture medium for 5 x 10 3 cells/well CT.4S cells for 48 h. They were then pulsed with [3H]thymidine at 1 /~Ci/well for 16 h and harvested. IL-6. The IL-6-dependent murine hybridoma cell line, B9, was used to titrate IL-6 levels [22]. Several triplicated dilutions of supernatants from LPS-activated spleen cells were added to the culture medium of 5 x l 0 3 cells/well B9 indicator cells, in RPMI supplemented with 5% FCS and 5 x 10-5 M 2-ME. After 48 h the cultures were pulsed with 1/~Ci/well of tritiated thymidine and harvested 18 h later. In all bioassays, the cells were harvested onto glass microfiber filter strips (Whatman GF/B) (Whatman, Maidstone, UK) by using a Brandel Cell Harvester (Gaithersburg, MD) which were counted for incorporation of tritiated thymidine with a Beckman liquid scintillation counter. 2.6. Statistical analysis Student's t-test was used to determine the significance of differences. A P value of at least 0.05 was considered statistically significant. Most of the significant values had P values <0.01. 2. 7. Detection of cell surface antigens TIP macrophages suspended in PBS (106 cells/0.1 ml) were incubated for 30 min at 4~ with antibodies to macrophage specific antigens, M l/70 (anti MAC-1), M3/38 (anti MAC-2) and M3/84 (anti MAC-3)] [23-25]. The antibodies were contained in hybridomas culture supernatants (American Type Culture Collection, Rockville, MD). The samples were washed twice with PBS Dulbecco's (containing 0.02% (w/v)
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sodium azide and 0.5% (w/v) bovine serum albumin (Sigma, St Louis, MO) and then incubated for 30 min at 4~ with fluorescein isothiocyanate (FITC) conjugated goat F(ab')2 anti rat IgG (Cappel, Malvern, PA). After washings, the cells were resuspended in PBS-sodium azide and kept on ice. The frequency and fluorescence profile were determined using the fluorescence-activated cell sorter (Elite, Coulter, Electronics, Hialeah, FL). The scatter gates were set to exclude erythrocytes, dead cells, most lymphocytes and polymorphonuclear leukocytes. A total of 10 000 viable cells were analyzed for fluorescence intensity.
2.8. Radiography Radiographs of tibia and femur were performed on a monolayered film Cronex MRF31 | (Dupont) using a Mammodiagnost | (Philips) fitted with a molybdenum anode 0.6 mm at a film focus distance of 55 cm. 2.9. Histology of bones After fixation in neutral buffered formalin, the tibias were decalcified in EDTA and embedded in paraffin. Frontal sections of the proximal end were stained with hematoxylin-eosin. 2.10. Light and electron microscopy of macrophages Samples of thioglycollate induced peritoneal macrophages were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 30 min. After several rinses in the same buffer containing 0.2 M sucrose, the samples were postfixed in 1% osmium tetroxide for 2 h at 4~ After dehydration in graded series of ethanol, the samples were processed through propylene oxide and embedded in Epon 812. Semi-thin sections were stained with methylene blue for light microscopy. Ultra-thin sections were stained successively with saturated aqueous uranyl acetate solution and 0.2% lead citrate. Examination was performed with a EM 109 Zeiss electron microscope at 80 kV. 3. Results
3.1. Production and secretion of interleukins by spleen cells IL-1. Reduced levels of IL-1 were observed in 6-week-old op/op mice. However, normal levels were found at 12 weeks and later (Fig. 1). IL-3. In 6-week-old op/op mutant mice, the level of IL-3 was greatly decreased (25% of normal). Interestingly, at the age of 22 weeks, important recovery of IL-3 level up to 50% of normal was observed (Fig. 2). IL-4. Greatly decreased (25% of normal) levels of IL-4 was observed in 6-week-old op/op mutant mice, whereas IL-4 level reached normal values at the age of 22 weeks (Fig. 3). IL-2 and IL-6. Whatever the age, the differences in the levels of IL-2 and IL-6 in mutant and normal mice were not statistically significant (P > 0.05) (Figs. 4 and 5). 3.2. Morphology of TIP macrophages At the age of 6 weeks, the average number of peritoneal macrophages yielded from
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op/op mice was decreased: 5 x 106 4- 2 • 106 vs 34 x 106 4- 5 • l0 b from normal mice, whereas at 22 weeks the average number of TIP op macrophages was similar to control values (25 x 106 4- 7 x 106). In normal mice, whatever the age, light microscopy of semi-thin sections evidenced that TIP macrophages appeared as an heterogeneous population considering the size, shape and appearance of granulations. In contrast, macrophages from op/op mice observed at 6 weeks of age constituted only one morphological type. They are uniform in size and contain large clear cytoplasmic vacuoles (Figs. 6a,b). When observed by electron microscopy, TIP macrophages from normal mice varied in sizes, and well-developed organelles were found in their cytoplasm (Fig. 7a). Scattered strands of rough endoplasmic reticulum and well-developed Golgi apparatus were observed as well as numerous large mitochondria, vesicles and lysosomes. Within the secondary lysosomes, ingested material was found at different stages of degradation. The surface of macrophages was ruffled as assessed by numerous microvilli and blebs of the cytoplasmic membrane. In contrast, TIP macrophages from 6-week-old op/op mice, were larger and uniform in size. They possessed much less developed organelles and the microvillous
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projections were poorly developed. In 80% of these macrophages, ingested spumous material was found in large secondary lysosomes occupying most of the cytoplasm (Fig. 7b). The proportion of macrophages with large secondary lysosomes was established by direct microscopic observations. At 22 weeks of age, the op/op macrophage population was more heterogeneous and the number of cells with large cytoplasmic inclusions decreased.
3.3. Expression of macrophage differentiation antigens on TIP macrophages Expression of macrophage surface differentiation antigens was studied at 6 and 22 weeks of age in op/op mutants and their normal littermates. The presence of 3 antigens was detected by immunofluorescence flow cytometry. The results are presented in Fig. 6. Levels of MAC-1 remained identical to control mice at different ages (Fig. 8a). At the age of 6 weeks, a reduction up to 50% was observed in the expression of MAC-2 antigen (a 32-kDa glycoprotein) in op/op mice. On the other hand, the expression of MAC-2 antigen reached normal levels at the age of 22 weeks (Fig. 8b). The level of MAC-3 (a 110-kDa glycoprotein), was sighltly decreased at 6 weeks and was similar to normal at 22 weeks (Fig. 8c).
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3.4. Radiographic and histologic examination of osteopetrotie bones at different ages X-ray examination of the tibia and femur was performed at 6, 12 and 22 weeks of age, Six-week-old op/op mice are osteopetrotic according to the classical definition: a poorly developed marrow cavity giving an uniformely opaque appearance of the long bones (Fig. 9). Histologic examination correlates an impaired development of the marrow cavity with endochondral bone trabeculae occupying both the metaphysis and diaphysis.
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Fig, 4. IL-2 production in normal and op/op mice at different ages. Dilutions of supernatants from ConA-activated spleen cells were assayed for IL-2 production using the CTLL indicator line. Results are expressed in counts/min. The mean value + S.D. of triplicate cultures is presented.
Cores of calcified cartilage still present in these trabeculae indicate deficient remodelling (Fig. 10). In contrast, at the age of 22 weeks, X-ray examination of the op/op long bones revealed radiolucent diaphysis, indicating partial bone resorption (Fig. 9c). Histologic examination (Fig. 10c) confirmed the presence of bone marrow. Such results indicate partial recovery with age of the osteopetrotic bone lesions.
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Fig. 5. IL-6 production in normal and op/op mice at different ages. Dilutions of supernatants from LPSactivated spleen cells were assayed for IL-6 production using the B9 indicator line. Results are expressed in counts/min. The mean value q- S.D. of triplicate cultures is presented.
4. Discussion
The op/op mutation in the mouse is attributed to a severe deficiency of mature osteoclasts and macrophages [17,26]. Both cell populations were suggested to originate from a common hemopoietic progenitor [27]. Udagawa et al. [15] evidenced that mature macrophages (including peritoneal cells) may differentiate into osteoclast-like cells under suitable environmental conditions. However, the cascade of events and signals that guide osteoclast development through precursor proliferation, chemotaxis and attachment to bone, fusion and differentiation, is far from being well-known. Recently, the involvment of M-CSF as a causal agent of osteopetrosis in this
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Fig. 6. Semi-thin sections of TIP macrophages from (a) normal and (b) osteopetrotic mice at 6 weeks of age (magnification x 240). (a) Macrophages from normal mice vary in sizes and cytoplasmic vacuoles of different sizes and densities are observed. (b) Macrophages from op mice are more uniform in size and contain large clear cytoplasmic vacuoles.
mutant has been pointed out. Cultured osteoblasts and fibroblasts from op/op mice do not produce and secrete M-CSF [12,13]. Daily administration of M-CSF to the newborn mutants corrects in vivo the impaired bone resorption [28]. The failure in M-CSF production has been related to a mutation within the M-CSF coding region on chromosome 3 [291.
Fig. 7. Electron microscopy of TIP macrophages from (a) normal and (b) osteopetrotic mice at 6 weeks of age (magnification x 2200). (a) Macrophages from normal mice vary in size and contain well-developed organelles. (b) Macrophages from op mice are larger and their cytoplasm contains large secondary lysosomes.
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Fig. 9. Radiographs of tibias: a, 6-week-old normal mouse; b, 6-week-old op/op mouse; c, 22-week-old op/op mouse. At 22 weeks, a radiolucent diaphysis is visible.
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Fig. 8. lmmunofluorescence flow cytometry analysis of TIP macrophages from normal and op/op mice at different ages. TIP macrophages were incubated with monoclonal antibodies directed to MAC-I, MAC-2 or MAC-3, followed by another incubation with FITC conjugated goat F(ab)'2 anti rat lgG. The histograms are plotted with the relative cell number on a linear scale and fluorescence on a logarithmic scale, a, MAC-I; b, MAC-2; c, MAC-3.
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Fig. 10. Decalcified sections of proximal tibias: a, 6-week-old normal mouse; b, 6-week-old op/op mouse; c, 22-week-old op/op mouse. At 22 weeks, partial bone resorption and hematopoietic areas are observed. Hematoxylin-eosin, magnification • 60.
The question of a possible involvment of different cytokines in the process of osteopetrosis is studied in the present work. Since a marrow cavity develops progressively in older mutants, the op/op mouse [17] appears as a model of special interest for studying the relationships between cytokines and bone remodelling. In the present report, interleukin secretions by op/op splenic cells are compared with that of normal littermates at 6 weeks of age when the bone has classical osteopetrotic appearance and at 22 weeks of age when recovery of the bone lesions is noticeable by X-ray examination. Production of both IL-1 and IL-4 are markedly reduced in 6-week-old op/op mice and reach normal levels at 22 weeks. Gowen and Mundy [30] demonstrated that IL-1, already known as a highly pleiotropic cytokine released from activated monocytes or macrophages, was able to stimulate bone resorption by osteoclasts. IL-4 is produced by activated T-cells. Its potential role in bone remodelling has been less investigated. Recently, IL-4 was suggested to play an important role on the inhibitory regulation of bone resorption [31]. Therefore, the partial healing of bone lesions in conjunction with the normalization of the levels of IL-1 and II-4 in the op/op mice may reflect the involvment of these factors in the regulation of bone cell activity. But these findings may also be related to the deficiency of M-CSF in these osteopetrotic mutants. Indeed, IL-1 could induce the proliferation of pluripotent progenitors in the bone marrow and substitute for CSF in human bone marrow cultures [32]. Recently, Fibbe et al. [33] have shown that IL-1 may lead to the production of M-CSF and G-CSF by human marrow stromal cells. Recent data showed that IL-4 may trigger the release of M-CSF or G-CSF from monocytes [34], and enhance the proliferation of granulocyte-macrophage-progenitors [35]. However, they are conflicting results concerning the role of CSFs and IL-3 in osteoclastic bone resorption. The results of Takahashi et al. [36] show that the culture system influences the CSFs effects on osteoclast development. In vivo, M-CSF appears to be an
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important factor for the growth of osteoclast progenitors and a deficiency of M-CSF results in osteopetrotic disorders in bone [12,13,26,28]. On the other hand, IL-3, also designed as multi-CSF, is severely decreased in 6week-old op/op mice but recover only partially at 22 weeks. IL-3, which was shown to induce differentiation (functional maturation) of the progeny of hemopoietic stem cells induces only partial maturation of the osteoclast [37]. The latter is completed by additional factors, such as 1,25(OH)2 D 3 o r M-CSF [38]. Thus, even if IL-3 reaches normal (or close to normal) levels during the op/op mouse lifespan, this normalization can not be expected to lead to a complete recovery of the osteopetrotic bone lesions. Expression of differentiation antigens is studied on op/op macrophage-subpopulations and differs from that of the normal mouse. MAC-2 and MAC-3 appear to be expressed only on the monocytic line at some stage after divergence from the granulocytic line, while MAC-1, which is associated with type 3 complement receptor, is found on both lineages [23-25]. We found that the expression of MAC-2 is markedly reduced in thioglycollateinduced macrophages from 6-week-old op/op mice. MAC-2 is known to be induced in macrophages only in response to specific differentiative signals, such as high inflammatory agents, especially thioglycollate [24,25]. Thus, MAC-2 is specific for a subpopulation in a distinct stage of differentiation [23,24]. Yet, the differentiation signals are not identified. The question whether or not they are related to the cytokines studied here is raised. We also evidenced (unpublished results) a reduced MAC-2 expression in macrophages from other murine osteopetrotic mutations: grey lethal (gl/gl) and microphtalmic (mi/mi). Thus, the possible involvement of macrophage MAC-2 differentiation antigen in the generation and/or the function of normal osteoclasts needs further investigation. On the other hand, the difference in the morphology of the macrophages observed in young op/op mice compared with normal littermates, could reflect a defect in intrinsic function or the influence of disturbed micro-environmental conditions. Variations in interleukin secretions, along with expression of macrophage specific antigen reported here, suggest that these factors could play a role in bone remodelling. A correlation seems to exist between the partial spontaneous recovery of bone resorption in this mutant and the normalization of some of their immunological functions. 5. Acknowledgements This work was supported by CNRS (Department of Life Sciences) and by the Association pour la Recherche contre le Cancer (ARC, Grant 6194). Dr E. Tzehoval was a foreign senior research fellow of CNRS (Department of Life Sciences). Part of this work was presented as poster (No. 568) at the Xlth International Conference on Calcium Regulating Hormones, Florence, Italy, April 24-29, 1992. 6. References 1 Albers-Sch6nberg H. Rontgenbilder einer seltenen Knochenerkrankung. Muench Med Wochenschr 1904;51:365.
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Marks SC Jr. Congenital osteopetrotic mutations as probes of the origin, structure and function of osteoclasts. Clin Orthop 1984;189:239-263. Walker DG. Osteopetrosis in mice cured by temporary parabiosis. Science 1973;180:875. Walker DG. Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells. Science 1975;190:784-785. Milhaud G, Labat ML, Graf B, Juster M, Balmain N, Moutier R, Toyama K. Demonstration cin6tique, radiographique et histologique de la gu~rison de l'ost~op~trose eong6nitale du rat. CR Acad Sci Paris S~r D 1975;280:2485-2488. Marks SC Jr. Osteopetrosis in the ia rat cured by spleen cells from a normal littermate. Am J Anat 1976;146:331-337. Marks Jr SC. Seifert MF, Mac Guire JL. Congenitally osteopetrotic mice are not cured by transplants of spleen or bone marrow cells from normal littermates. Metab Bone Dis Rel Res 1984;5:183-186. Marks Jr SC. Osteopetrosis in the toothless (tl) rat: presence of osteoclasts but failure to respond to parathyroid extract or to be cured by infusion of spleen or bone marrow cells from normal littermates. Am J Anat 1977;149:289-297. Labat ML, Milhaud G. Osteopetrosis and the immune deficiency syndrome. J Bone Miner Res 1986;4:131-212. Milhaud G, Labat ML, Parant M, Damais C, Chedid T. Immunological defect and its correction in the osteopetrotic mutant rat. Proc Natl Acad Sci USA 1977;74:339-342. Labat ML, Tzehoval E, Moricard Y, Feldman M, Milhaud G. Lack of a T-cell dependent subpopulation of macrophages in (dichloromethylene) diphosphonate treated mice. Biomed Pharmacother 1983;37:270-276. Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW Jr, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER. Total absence of colony stimulating factor 1 in the macrophage deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 1990;87:4828-4832. Felix R, Cecchini MG, Hofstetter W, Elford RR, Stutzer T, Fleisch H. Impairment of macrophage colony stimulating factor production and lack of resident bone marrow macrophages in the osteopetrotic op/op mouse. J Bone Min Res 1990;5:781-789. Metcalf D. The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood 1986;67:257-267. Udagawa N, Tokahashi N, Akatsu T, Tanaka H, Sasaki T, Nishiara T, Koga T, Martin TJ, Suda T. Origin of osteoclats: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow derived stromal cells. Proc Natl Acad Sci USA 1990;87:7260-7264. Hagenaars CE, van der Kraan AAM, Kawilarang-de Haas EWN, Visser JWN, Nijwelde PJ. Osteoclast formation from cloned pluripotent hemopoietic stem ceils. J Bone Min Res 1989;6:179-189. Marks SC Jr, Lane PW. Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. J Hered 1976;67:11-18. Coulon PJ. A rapid biologic assay for the detection of interleukin-1. J Immunol 1983; 131:1280-1282. Gillis S, Ferm MM, Ou W, Smith KA. T cell growth factor: parameters of production and a quantitative microassay for activity. J lmmunol 1978;120:2027-2032. Nabel G, Galli J J, Dvorak AM, Dvorak HF, Cantor H. Inducer T lymphocytes synthetize a factor that stimulates proliferation of cloned mast cells. Nature 1981;291:332-333. Hu-Li J, Ohara J, Watson C, Tsang W, Paul WE. Derivation of a T cell line that is highly responsive to IL-4 and IL-2 (CT.4R) and of an IL-2 hyporesponsive mutant of that line (CT.4S). J lmmunol 1989;142:800-807. Helle M, Boeije L, Aarclan LA. Functional discrimination between interleukin 6 and interleukin 1. Eur J lmmunol 1988;18:1535-1540. Springer TA, Galfre G, Secher DS, Milstein C. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur J lmmunol 1978;8:539-551. Ho MK, Springer TA. Mac-2, a novel 32 000 MR, mouse macrophage subpopulation-specific antigen defined by monoclonal antibodies. J Immunol 1982;128:1221-1228. Springer TA. Monoclonal antibody analysis of complex biological system. J Biol Chem 1981;256:3833-3839.
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Wiktor-Jedrzejczak W, Ahmed A, Szczylik C, Skelly R. Hematological caracterization of congenital osteopetrosis in op/op mouse. Possible mechanism for abnormal macrophage differentiation. J Exp Med 1982;156:1516-1527. Schneider GB, Relfson M, Nicolas S. Pluripotent hemopoietic stem cells give rise to osteoclasts. Am J Anat 1986;177:505-511. Felix R, Cecchini MG, Fleisch H. Macrophage stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 1990;127:2592-2594. Yoshida H, Hayashi SI, Kunisada T, Ogawa M, Nishikawa S, Okumura H, Sudo T, Shultz LD, Nishikawa SI. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990;345:442-443. Gowen M, Mundy GR. Actions of recombinant interleukin-1, interleukin-2 and interferon 3, on bone resorption in vitro. J lmmunol 1986;136:2478-2482. Watanabe K, Tanaka Y, Morimoto I, Yahata K, Zeki K, Fujihira T, Yamashita U, Eto S. Interleukin-4 as a potent inhibitor of bone resorption. Biochem Biophys Res Commun 1990;172:1035-1041. van Damme J, De Ley M, Opdenakker G, Billiau A, De Somer PD. Homogeneous interferon inducing 22 k factor is related to endogenous pyrogen and interleukin-l. Nature 1985;314:266-268. Fibbe WE, van Damme J, Billiau A, Goselink HM, Voogt PJ, van Eeden G, Ralph P, Altrock BW, Falkenburg JHF. lnterleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor. Blood 1988;71:430-435. Wieser M, Bonifer R, Oster W, Lindemann A, Mertelsmann R, Herrmann F. Interleukin-4 induces secretion of CSF for granulocytes and CSF for macrophages by peripheral blood monocytes. Blood 1989;73:1105-1108. Peschel C, Paul WB, Ohara J, Green I. Effects of B cell stimulatory factor-l/interleukin 4 on hematopoietic progenitor cells. Blood 1987;708:254-263. Takahashi N, Udagawa N, Akatsu T, Tanaka H, Shionome M, Suda T. Role of colony-stimulating factors in osteoclast devlopment. J Bone Miner Res 1991;6:977-985. Sonada Y, Yang YC, Wong GG, Clark SC, Ogawa M. Analysis in serum-free culture of the targets of recombinant human hemopoietic growth factors: interleukin 3 and granulocyte/macrophage colony stimulating factor are specific for early developmental stages. Proc Natl Acad Sci USA 1988;85:4360-4364. Hattersley G, Chambers TJ. Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue. J Cell Physiol 1990;142:201-209.
Bone and Mineral 23 (1993) 317-332
Immune cell defects affect bone remodelling in osteopetrotic op/op mice C. Philippart a, E. Tzehoval b, Y. Moricard a, A.-F. Bringuier a, C. Seebold a, F.-M. Lemoine c, A. Arys d, N. Dourov d, M.-L. Labat *a aCNRS, Laboratoire de Physiopathologie Osseuse, Centre de Recherches Biom~dicales des Cordeliers, 15 Rue de l'Ecole de Medecine, 75270 Paris Cedex 06, France hDepartment of Cell Biology, The Weismann Institute of Science, Rehovot, Israel CDEpartement d'hEmatologie, HSpital St-Antoine, 75012 Paris, France dLaboratoire d'Anatomie Pathologique et de Microscopie Electronique, Facult~ de MEdecine, Universit~ Libre de Bruxelles, Campus Erasme B6t. C, 808 route de Lennick, B-1070 Brussels, Belgium
(Received 4 May 1993; revision received 5 August 1993; accepted 1 September 1993)
Abstract The aim of the present work was to further characterize immunological defects in osteopetrosis. The op/op mutant mouse is of particular interest since a marrow cavity develops spontaneously in older animals. The interleukin production (IL-I, IL-2, IL-3, IL-4, IL-6), the presence of macrophage differentiation antigens and the evolution of the bone lesions were studied in osteopetrotic and normal mice. Low levels of IL-1, IL-3 and IL-4 were observed at the age of 6 weeks in the op/op mice. However, at 22 weeks of age, the level of IL-1 and IL-4 returned to normal value in these op/op mice whereas the level of IL-3 remained partially decreased at the same age. Furthermore, macrophage expression of MAC-2 antigen, reduced at 12 weeks of age was found to be normal 10 weeks later. These immunological defects and their recovery seems to be concomitant with the healing of the bone lesions. Key words: Osteopetrosis; Interleukins; Macrophages
1. Introduction Osteopetrosis is characterized by a uniformly dense appearance of most of all long bones at X-ray examination [1]. This is due to a failure in development o f the mar* Corresponding author. 0169-6009/93/$06.00 9 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0169-6009(93)00636-H
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row cavity as a result of deficient osteoclastic bone resorption [2]. Two main causes of deficient bone resorption are recognized; either the defect lies in hematopoietic stem cells with osteolytic capabilities or in the microenvironment, unable to provide appropriate signals for mononuclear cells proliferation and/or differentiation into osteoclasts [3-8]. Following the demonstration by Walker in 1973 [3] of the hematopoietic origin of the disease, further studies by Labat and coworkers led to reconsideration of osteopetrosis as an immune disease [9,10]. Osteopetrosis, either inherited as in the op/op mutant rat or experimentally induced by a biphosphonate treatment, was associated with an early atrophy of the thymus and an impairment of T-cell functions [11]. A single bone marrow injection cured the immune deficiency and the bone lesions of the osteopetrotic rat [5]. However, op/op mice could not be cured by such treatment, suggesting that the defect lies in the bone marrow environment [7]. Indeed, it was shown that cultured osteoblasts and fibroblasts from op/op mice did not produce and secrete macrophage colony-stimulating factor (M-CSF) [12,13]. MCSF is necessary for the growth and differentiation of mononuclear phagocytes [14] which are considered to be osteoclast precursors [15]. It was suggested that M-CSF cooperates with other cytokines, such as IL-3 and IL-1, in the formation of osteoclasts [16]. Thus, the op/op mouse could serve as an excellent model to study the biology of interleukins in relation to bone resorption and to macrophage differentiation. In this study, we report that besides defective production of M-CSF documented by Wiktor-Jedrzejczak et al. [12] and Felix et al. [13], interleukin secretions are impaired in op/op mice. Low levels of IL-1, IL-3 and IL-4 are observed at the age of 6 weeks. Between 12 and 22 weeks, IL-1 and IL-4 reach almost normal levels, whereas IL-3 remains partially decreased at 22 weeks of age. Furthermore, in older mutants, we observe normalisation in the op macrophage ultrastructure and phenotype. On the other hand, the development of long bone marrow cavities is also noted. We can not assert that the increased cytokine levels cause increased osteoclast activity. Nevertheless, normal levels of cytokines and changes in ultrastucture and phenotype of macrophage are concomitant with the healing of bone lesions. These results suggest that increased levels of IL-1, IL-3 and IL-4 may not only reflect a change in macrophage-modulating activity but that these cytokines may also be involved in bone remodelling. 2. Materials and methods 2.1. Mice
Osteopetrotic (op/+) heterozygotes-tested breeding pairs were purchased from the Jackson Laboratory (Bar Harbour, ME). Animals were maintained in controled environment modules (ESI-Flufrance Cachan, France). Mating heterozygotes results in the production of op/op and normal littermates (+/+ or +/op). The op/op mutant mice are recognized by the absence of incisor teeth as soon as 7 days after birth [17]. After weaning, animals were fed on a standard sterile diet RO3-40 (UAR Villemoisson Orge, France) given as a powder to the mutants. Four mutant mice (op/op) and 4 controls (including +/+ and +/op genotypes) were individually tested in each age group: 6, 12 and 22 weeks.
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2.2. Reagents The following culture media or reagents were used throughout the experiments. Thioglycollate broth was prepared according to the manufacturer's instructions (Difco, Detroit, MI). Culture media such as PBS Dulbecco's, RPMI 1640, DMEM and fetal calf serum were purchased from Gibco (Grand Island, NY). Heparin: thromboliquine was from Organon Teknika BV (Boxtel, The Netherlands). Mitogens such as lipopolysaccharide (LPS) from E. coli 0111 :B4 (Sigma, St-Louis, MO), concanavalin A (ConA) (ICN Immunobiologicals, Lisle, IL) and phytohemagglutinin (PHA) (WeUcome, Dartford, UK) were used. Cells were pulsed with tritiated thymidine, specific activity 23 Ci/mmol (Dositek, Orsay, France). All cell cultures were performed in 24- or 96-well microplates (Costar, Cambridge, MA). Specificity for each bioassay was tested using recombinant interleukins and antiinterleukins (Genzyme, Boston, MA). Cell lines used in the bioassays were from ATCC for LBRM-33-1A5 and CTLL.B9 and CT.4S were a generous gift from W. Paul (NIH) and MC/9 was kindly provided by the Weizmann Institute of Science. 2.3. Thioglycollate induced peritoneal macrophages (TIP macrophages) Mice were injected intraperitoneally (i.p.) with 3 ml of thioglycollate broth. Four days later, the mice were killed and injected i.p. with 10 ml of PBS Dulbecco's containing 5 units heparin/ml. The peritoneum was gently massaged and the peritoneal fluid was aspirated with a 10 ml syringe. The cell suspensions were centrifuged at 4~ at 200 x g for 10 min. The supernatant was discarded and the cells were resuspended in PBS. Cell preparation consistently contained > 90% macrophages, of which more than 90% were viable cells (Trepan blue die exclusion). 2.4. Spleen cells Spleens, once aseptically removed from op/op or normal littermates at different ages, were flushed by injecting PBS into the organ. The recovered cells were then seeded in 24-well microplates at the concentration of 10 7 cells/well. Production of IL-1 and IL-6 was determined following overnight (18 h) culture in DMEM supplemented with 40#g/ml gentamycin in the presence of 30#g/ml LPS. Production of IL-2, IL-3 and IL-4 was determined following overnight (18 h) culture in RPMI 1640 supplemented with 10% FCS, 5 x 10-5 M 2-mercaptoethanol (2-ME) and 40#g/ml gentamycin in presence of 10#g/ml ConA. The supernatants were centrifuged at 900 x g for 10 min, then serially diluted and assayed in triplicates for interleukins. 2.5. Bioassays of interleukins Supernatants prepared from in vitro cultures of mitogen-activated spleen cells were bioassayed for the following interleukins: IL-1, IL-2, IL-3, IL-4 and IL-6, using selected responsive cell lines. Experiments were all performed in 96-well microplates. Each well contained a constant amount of diluted supernatant (100#1) and I00#1 of cells. Recombinant cytokines were used as positive controls for bioassays. For each bioassay, the specificity was tested using antibodies directed against the cytokine study. The cytokine linked to its specific antibody is unable to stimulate cell proliferation. IL-1. A thymoma cell line LBRM-33-1A5, was used to assay for IL-1, based on the ability of IL-1 to augment IL-2 production as a costimulant with PHA [18].
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Several triplicated dilutions of supernatants from LPS-activated spleen cells were added to the culture medium of 5 x 10 4 cells/well LBRM-33-1A5 in presence of PHA at a final concentration of 3#l/ml. After 18 h, IL-2 activity was determined by removing 100/~l samples from the wells and incubating them with 100 ttl of IL-2dependent continuous T lymphocytes line, CTLL (5 x 10 3 cells/well) [19] for an additional 20 h, followed by a 12-h pulse with 1/~Ci/well of tritiated thymidine and harvested. An IL-2 standard mouse factor, prepared from ConA-stimulated mouse spleen cells, was used as a positive control. Results were expressed in counts/rain. IL-2. Proliferation of the IL-2-dependent CTLL indicator cells was determined as described previously [19]. Briefly, triplicated dilutions of supernatants from ConAactivated spleen cells were added to the culture medium of 5 x 10 3 cells/well CTLL indicator cells, in RPMI supplemented with 10% FCS and 5 • 10-5 M 2-ME. After 20 h the cultures were pulsed (12 h) with 1/~Ci/well of tritiated thymidine and harvested. IL-3. Several triplicated dilutions of supernatants from ConA-activated spleen cells were added to the culture medium of 5 • 10 3 cells/well MC/9 indicator cells [20], in DMEM supplemented with 10% FCS and 5 • 10-5 M 2-ME. After 48 h the cultures were pulsed (18 h) with 1 t~Ci/well of tritiated thymidine and harvested. IL-4. The highly responsive CT.4S cell line was used to assay IL-4 activity in ConA-activated spleen cell cultures [21]. Several triplicated dilutions of the supernatant in RPMI supplemented with 10% FCS and 5 x 10-5 M 2-ME and 0.4 mg/ml gentamycine, were used as culture medium for 5 x 10 3 cells/well CT.4S cells for 48 h. They were then pulsed with [3H]thymidine at 1 /~Ci/well for 16 h and harvested. IL-6. The IL-6-dependent murine hybridoma cell line, B9, was used to titrate IL-6 levels [22]. Several triplicated dilutions of supernatants from LPS-activated spleen cells were added to the culture medium of 5 x l 0 3 cells/well B9 indicator cells, in RPMI supplemented with 5% FCS and 5 x 10-5 M 2-ME. After 48 h the cultures were pulsed with 1/~Ci/well of tritiated thymidine and harvested 18 h later. In all bioassays, the cells were harvested onto glass microfiber filter strips (Whatman GF/B) (Whatman, Maidstone, UK) by using a Brandel Cell Harvester (Gaithersburg, MD) which were counted for incorporation of tritiated thymidine with a Beckman liquid scintillation counter. 2.6. Statistical analysis Student's t-test was used to determine the significance of differences. A P value of at least 0.05 was considered statistically significant. Most of the significant values had P values <0.01. 2. 7. Detection of cell surface antigens TIP macrophages suspended in PBS (106 cells/0.1 ml) were incubated for 30 min at 4~ with antibodies to macrophage specific antigens, M l/70 (anti MAC-1), M3/38 (anti MAC-2) and M3/84 (anti MAC-3)] [23-25]. The antibodies were contained in hybridomas culture supernatants (American Type Culture Collection, Rockville, MD). The samples were washed twice with PBS Dulbecco's (containing 0.02% (w/v)
C. Philippart et al./ Bone Miner. 23 (1993) 317-332
321
sodium azide and 0.5% (w/v) bovine serum albumin (Sigma, St Louis, MO) and then incubated for 30 min at 4~ with fluorescein isothiocyanate (FITC) conjugated goat F(ab')2 anti rat IgG (Cappel, Malvern, PA). After washings, the cells were resuspended in PBS-sodium azide and kept on ice. The frequency and fluorescence profile were determined using the fluorescence-activated cell sorter (Elite, Coulter, Electronics, Hialeah, FL). The scatter gates were set to exclude erythrocytes, dead cells, most lymphocytes and polymorphonuclear leukocytes. A total of 10 000 viable cells were analyzed for fluorescence intensity.
2.8. Radiography Radiographs of tibia and femur were performed on a monolayered film Cronex MRF31 | (Dupont) using a Mammodiagnost | (Philips) fitted with a molybdenum anode 0.6 mm at a film focus distance of 55 cm. 2.9. Histology of bones After fixation in neutral buffered formalin, the tibias were decalcified in EDTA and embedded in paraffin. Frontal sections of the proximal end were stained with hematoxylin-eosin. 2.10. Light and electron microscopy of macrophages Samples of thioglycollate induced peritoneal macrophages were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 30 min. After several rinses in the same buffer containing 0.2 M sucrose, the samples were postfixed in 1% osmium tetroxide for 2 h at 4~ After dehydration in graded series of ethanol, the samples were processed through propylene oxide and embedded in Epon 812. Semi-thin sections were stained with methylene blue for light microscopy. Ultra-thin sections were stained successively with saturated aqueous uranyl acetate solution and 0.2% lead citrate. Examination was performed with a EM 109 Zeiss electron microscope at 80 kV. 3. Results
3.1. Production and secretion of interleukins by spleen cells IL-1. Reduced levels of IL-1 were observed in 6-week-old op/op mice. However, normal levels were found at 12 weeks and later (Fig. 1). IL-3. In 6-week-old op/op mutant mice, the level of IL-3 was greatly decreased (25% of normal). Interestingly, at the age of 22 weeks, important recovery of IL-3 level up to 50% of normal was observed (Fig. 2). IL-4. Greatly decreased (25% of normal) levels of IL-4 was observed in 6-week-old op/op mutant mice, whereas IL-4 level reached normal values at the age of 22 weeks (Fig. 3). IL-2 and IL-6. Whatever the age, the differences in the levels of IL-2 and IL-6 in mutant and normal mice were not statistically significant (P > 0.05) (Figs. 4 and 5). 3.2. Morphology of TIP macrophages At the age of 6 weeks, the average number of peritoneal macrophages yielded from
322
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op/op mice was decreased: 5 x 106 4- 2 • 106 vs 34 x 106 4- 5 • l0 b from normal mice, whereas at 22 weeks the average number of TIP op macrophages was similar to control values (25 x 106 4- 7 x 106). In normal mice, whatever the age, light microscopy of semi-thin sections evidenced that TIP macrophages appeared as an heterogeneous population considering the size, shape and appearance of granulations. In contrast, macrophages from op/op mice observed at 6 weeks of age constituted only one morphological type. They are uniform in size and contain large clear cytoplasmic vacuoles (Figs. 6a,b). When observed by electron microscopy, TIP macrophages from normal mice varied in sizes, and well-developed organelles were found in their cytoplasm (Fig. 7a). Scattered strands of rough endoplasmic reticulum and well-developed Golgi apparatus were observed as well as numerous large mitochondria, vesicles and lysosomes. Within the secondary lysosomes, ingested material was found at different stages of degradation. The surface of macrophages was ruffled as assessed by numerous microvilli and blebs of the cytoplasmic membrane. In contrast, TIP macrophages from 6-week-old op/op mice, were larger and uniform in size. They possessed much less developed organelles and the microvillous
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projections were poorly developed. In 80% of these macrophages, ingested spumous material was found in large secondary lysosomes occupying most of the cytoplasm (Fig. 7b). The proportion of macrophages with large secondary lysosomes was established by direct microscopic observations. At 22 weeks of age, the op/op macrophage population was more heterogeneous and the number of cells with large cytoplasmic inclusions decreased.
3.3. Expression of macrophage differentiation antigens on TIP macrophages Expression of macrophage surface differentiation antigens was studied at 6 and 22 weeks of age in op/op mutants and their normal littermates. The presence of 3 antigens was detected by immunofluorescence flow cytometry. The results are presented in Fig. 6. Levels of MAC-1 remained identical to control mice at different ages (Fig. 8a). At the age of 6 weeks, a reduction up to 50% was observed in the expression of MAC-2 antigen (a 32-kDa glycoprotein) in op/op mice. On the other hand, the expression of MAC-2 antigen reached normal levels at the age of 22 weeks (Fig. 8b). The level of MAC-3 (a 110-kDa glycoprotein), was sighltly decreased at 6 weeks and was similar to normal at 22 weeks (Fig. 8c).
C. Phihppart et al./ Bone Mzner, 23 (1993) 317-332
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3.4. Radiographic and histologic examination of osteopetrotie bones at different ages X-ray examination of the tibia and femur was performed at 6, 12 and 22 weeks of age, Six-week-old op/op mice are osteopetrotic according to the classical definition: a poorly developed marrow cavity giving an uniformely opaque appearance of the long bones (Fig. 9). Histologic examination correlates an impaired development of the marrow cavity with endochondral bone trabeculae occupying both the metaphysis and diaphysis.
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Cores of calcified cartilage still present in these trabeculae indicate deficient remodelling (Fig. 10). In contrast, at the age of 22 weeks, X-ray examination of the op/op long bones revealed radiolucent diaphysis, indicating partial bone resorption (Fig. 9c). Histologic examination (Fig. 10c) confirmed the presence of bone marrow. Such results indicate partial recovery with age of the osteopetrotic bone lesions.
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4. Discussion
The op/op mutation in the mouse is attributed to a severe deficiency of mature osteoclasts and macrophages [17,26]. Both cell populations were suggested to originate from a common hemopoietic progenitor [27]. Udagawa et al. [15] evidenced that mature macrophages (including peritoneal cells) may differentiate into osteoclast-like cells under suitable environmental conditions. However, the cascade of events and signals that guide osteoclast development through precursor proliferation, chemotaxis and attachment to bone, fusion and differentiation, is far from being well-known. Recently, the involvment of M-CSF as a causal agent of osteopetrosis in this
C Philippart et al./Bone Miner. 23 (1993) 317-332
327
Fig. 6. Semi-thin sections of TIP macrophages from (a) normal and (b) osteopetrotic mice at 6 weeks of age (magnification x 240). (a) Macrophages from normal mice vary in sizes and cytoplasmic vacuoles of different sizes and densities are observed. (b) Macrophages from op mice are more uniform in size and contain large clear cytoplasmic vacuoles.
mutant has been pointed out. Cultured osteoblasts and fibroblasts from op/op mice do not produce and secrete M-CSF [12,13]. Daily administration of M-CSF to the newborn mutants corrects in vivo the impaired bone resorption [28]. The failure in M-CSF production has been related to a mutation within the M-CSF coding region on chromosome 3 [291.
Fig. 7. Electron microscopy of TIP macrophages from (a) normal and (b) osteopetrotic mice at 6 weeks of age (magnification x 2200). (a) Macrophages from normal mice vary in size and contain well-developed organelles. (b) Macrophages from op mice are larger and their cytoplasm contains large secondary lysosomes.
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328
Fig. 9. Radiographs of tibias: a, 6-week-old normal mouse; b, 6-week-old op/op mouse; c, 22-week-old op/op mouse. At 22 weeks, a radiolucent diaphysis is visible.
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22weeksI Intensity
Fig. 8. lmmunofluorescence flow cytometry analysis of TIP macrophages from normal and op/op mice at different ages. TIP macrophages were incubated with monoclonal antibodies directed to MAC-I, MAC-2 or MAC-3, followed by another incubation with FITC conjugated goat F(ab)'2 anti rat lgG. The histograms are plotted with the relative cell number on a linear scale and fluorescence on a logarithmic scale, a, MAC-I; b, MAC-2; c, MAC-3.
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Fig. 10. Decalcified sections of proximal tibias: a, 6-week-old normal mouse; b, 6-week-old op/op mouse; c, 22-week-old op/op mouse. At 22 weeks, partial bone resorption and hematopoietic areas are observed. Hematoxylin-eosin, magnification • 60.
The question of a possible involvment of different cytokines in the process of osteopetrosis is studied in the present work. Since a marrow cavity develops progressively in older mutants, the op/op mouse [17] appears as a model of special interest for studying the relationships between cytokines and bone remodelling. In the present report, interleukin secretions by op/op splenic cells are compared with that of normal littermates at 6 weeks of age when the bone has classical osteopetrotic appearance and at 22 weeks of age when recovery of the bone lesions is noticeable by X-ray examination. Production of both IL-1 and IL-4 are markedly reduced in 6-week-old op/op mice and reach normal levels at 22 weeks. Gowen and Mundy [30] demonstrated that IL-1, already known as a highly pleiotropic cytokine released from activated monocytes or macrophages, was able to stimulate bone resorption by osteoclasts. IL-4 is produced by activated T-cells. Its potential role in bone remodelling has been less investigated. Recently, IL-4 was suggested to play an important role on the inhibitory regulation of bone resorption [31]. Therefore, the partial healing of bone lesions in conjunction with the normalization of the levels of IL-1 and II-4 in the op/op mice may reflect the involvment of these factors in the regulation of bone cell activity. But these findings may also be related to the deficiency of M-CSF in these osteopetrotic mutants. Indeed, IL-1 could induce the proliferation of pluripotent progenitors in the bone marrow and substitute for CSF in human bone marrow cultures [32]. Recently, Fibbe et al. [33] have shown that IL-1 may lead to the production of M-CSF and G-CSF by human marrow stromal cells. Recent data showed that IL-4 may trigger the release of M-CSF or G-CSF from monocytes [34], and enhance the proliferation of granulocyte-macrophage-progenitors [35]. However, they are conflicting results concerning the role of CSFs and IL-3 in osteoclastic bone resorption. The results of Takahashi et al. [36] show that the culture system influences the CSFs effects on osteoclast development. In vivo, M-CSF appears to be an
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important factor for the growth of osteoclast progenitors and a deficiency of M-CSF results in osteopetrotic disorders in bone [12,13,26,28]. On the other hand, IL-3, also designed as multi-CSF, is severely decreased in 6week-old op/op mice but recover only partially at 22 weeks. IL-3, which was shown to induce differentiation (functional maturation) of the progeny of hemopoietic stem cells induces only partial maturation of the osteoclast [37]. The latter is completed by additional factors, such as 1,25(OH)2 D 3 o r M-CSF [38]. Thus, even if IL-3 reaches normal (or close to normal) levels during the op/op mouse lifespan, this normalization can not be expected to lead to a complete recovery of the osteopetrotic bone lesions. Expression of differentiation antigens is studied on op/op macrophage-subpopulations and differs from that of the normal mouse. MAC-2 and MAC-3 appear to be expressed only on the monocytic line at some stage after divergence from the granulocytic line, while MAC-1, which is associated with type 3 complement receptor, is found on both lineages [23-25]. We found that the expression of MAC-2 is markedly reduced in thioglycollateinduced macrophages from 6-week-old op/op mice. MAC-2 is known to be induced in macrophages only in response to specific differentiative signals, such as high inflammatory agents, especially thioglycollate [24,25]. Thus, MAC-2 is specific for a subpopulation in a distinct stage of differentiation [23,24]. Yet, the differentiation signals are not identified. The question whether or not they are related to the cytokines studied here is raised. We also evidenced (unpublished results) a reduced MAC-2 expression in macrophages from other murine osteopetrotic mutations: grey lethal (gl/gl) and microphtalmic (mi/mi). Thus, the possible involvement of macrophage MAC-2 differentiation antigen in the generation and/or the function of normal osteoclasts needs further investigation. On the other hand, the difference in the morphology of the macrophages observed in young op/op mice compared with normal littermates, could reflect a defect in intrinsic function or the influence of disturbed micro-environmental conditions. Variations in interleukin secretions, along with expression of macrophage specific antigen reported here, suggest that these factors could play a role in bone remodelling. A correlation seems to exist between the partial spontaneous recovery of bone resorption in this mutant and the normalization of some of their immunological functions. 5. Acknowledgements This work was supported by CNRS (Department of Life Sciences) and by the Association pour la Recherche contre le Cancer (ARC, Grant 6194). Dr E. Tzehoval was a foreign senior research fellow of CNRS (Department of Life Sciences). Part of this work was presented as poster (No. 568) at the Xlth International Conference on Calcium Regulating Hormones, Florence, Italy, April 24-29, 1992. 6. References 1 Albers-Sch6nberg H. Rontgenbilder einer seltenen Knochenerkrankung. Muench Med Wochenschr 1904;51:365.
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