Studies of the mechanism of spleen cell cure for osteopetrosis in ia rats: appearance of osteoclasts with ruffled borders

After ia (osteopetrotic) rats receive whole body radiation and an injection of spleen cells from a normal littermate, the dense, sclerotic skeleton characteristic of osteopetrosis is rapidly remodeled and becomes normal in appearance radiographically and histologically within three weeks. The mechanism of this skeletal transformation has been explored in cured ia rats by light and electron microscopic examination of osteoclasts. In ia rats less than 25 days of age, osteoclasts viewed by electron microscopy lack a ruffled border - the extensive elaboration of plasma membrane next to the bone surface. Cured ia rats have osteoclasts with ruffled borders indistinguishable from those of normal littermates. In ia rats that receive only 600 rads whole body radiation, osteoclasts are still present three weeks later, but appear abnormal by light microscopy, with dense nuclei and lacking cytoplasmic vacuoles next to the bone surface. Cured ia rats have two types of osteoclasts, one type indistinguishable from osteoclasts of normal littermates by light microscopy, the other resembling osteoclasts of ia rats that received radiation only. These data indicate that the mechanism of the spleen cell cure for osteopetrosis in ia rats is rapid remodeling of the skeleton produced by osteoclasts with ruffled borders. Whether normal spleen cells produce these osteoclasts directly by cell division or indirectly by elaboration of some unknown local factor required for formations of ruffled borders by ia osteoclasts is not known.     

Congenitally osteosclerotic (os/os) rabbits are not cured by bone marrow transplantation from normal littermates.

The osteopetrotic (os) rabbit is a lethal mutation of autosomal recessive inheritance characterized by hypocalcemia, hypophosphatemia, fibrosis of marrow spaces, and ultrastructural abnormalities in both osteoclasts and osteoblasts. Procedures involving the transplantation of cells from normal hemopoietic tissues, which are sources of osteoclast precursors, are known to cure osteopetrosis in several mutations including some children. We tested the ability of transplanted bone marrow and/or spleen from normal littermates to reverse the skeletal sclerosis in os rabbits. Treatment of 15 neonatal mutants consisted of immunosuppression by whole-body irradiation followed by transplantation of normal bone marrow and/or spleen cell suspensions. This treatment failed to prolong life span or to cure osteopetrosis judged radiographically and histologically for up to 3 weeks posttreatment, the longest time of survival. These data indicate that transplantation of stem cells from multiple hemopoietic tissues, procedures known to cure osteopetrosis in other mutations, is not effective in the os rabbit. These results support the hypothesis that the skeletal microenvironment is not capable of supporting the development and function of normal osteoclasts in this mutation.     

Osteopetrosis in the toothless (t1) 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.

Osteoclast have been observe for the first time in toothless (t1) rats, a mutation with inherits osteopetrosis as an autosomal recessive. The ability of t1 rats to raise the serum calcium concentration after injection of parathyroid extract was severely limited when compared with normal littermates. In addition, osteopetrosis in t1 rats is not cured by radiation and infusion of normal spleen or bone marrow cells from normal littermates, a method know to cure osteopetrosis in mutants of this and other species. This indirect evidence for a reduction in bone resorption as a cause of osteopetrosis in this mutation and the failure of transplanted cells to cure the disease are discussed in relation to the development and function of osteoclasts.     

Cellular specificity of the cure for neonatal osteopetrosis in the ia rat

Osteopetrosis in the ia/ia rat is known to be the result of reduced bone resorption due to abnormal osteoclasts. Studies in this mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia/ia rats. This investigation was pursued in an attempt to determine the cellular source of this cure by transplanting various populations of cells from 21-day-old normal rats to unrelated newborn ia/ia recipients. The effects of treatment were evaluated radiographically and by measuring the size of the tibial marrow cavity. The cellular suspensions that were effective in curing the disease were the Ficoll-Hypaque isolate of spleen, bone marrow, and newborn livers. The Ficoll-Hypaque isolates of lymph node, thymus, and blood, and the adherent pool of peritoneal cells and splenic cells did not produce a cure in the ia/ia recipients. These results suggest that the cellular source of the cure is a stem cell. This conclusion is further substantiated by the finding that Thy 1.1 antigen (a stem cell marker in the rat) is expressed on a majority of the cells from the donor sources that affected a cure.     

Defective osteoclast differentiation and function in the osteopetrotic (os) rabbit

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagues, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrate-resistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors--absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.     

Calcitonin inhibits accumulation of cyclic AMP in stimulated peritoneal macrophages from normal rats but not from osteopetrotic (incisors-absent) littermates

Macrophages and osteoclasts derive from related cell lines. In osteopetrotic mutants the function of osteoclasts is greatly reduced compared to that in normal animals or children and macrophage function is variably affected depending upon the mutation. To further explore macrophage function in osteopetrosis we examined the regulation of cyclic AMP production in macrophages from mutants and normal littermates of the osteopetrotic stock incisors-absent (ia) in the rat. Surface stimulation by latex particles of elicited peritoneal macrophages from normal or osteopetrotic (ia) mutant rats caused an identical increase in the accumulation of cyclic AMP. This effect was inhibited in normal animals by coincubation of macrophages with calcitonin (CT) but this inhibition was either absent or less marked in macrophages from mutant littermates. In contrast to human monocytes preincubation of rat macrophages with pertussis toxin did not relieve this inhibition. This implies that rat peritoneal macrophages respond to CT by a different mechanism. These results demonstrate altered macrophage function in osteopetrotic animals and may be functionally related to the reduced CT binding previously described in ia osteoclasts. Furthermore, the coexistence of reduced function of macrophages and osteoclasts in the ia mutation suggests that macrophages and osteoclasts share a common progenitor.     

Mechanical properties in long bones of rat osteopetrotic mutations.

Osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. Experimental studies with rat osteopetrotic mutations have shown increased bone density and decreased bone strength. However, it is not known if this reduction in bone strength is only due to changes in structure and geometry or if the tissue properties of bone material itself are changed as well. We have evaluated bone tissue properties with nanoindentation in three osteopetrotic mutations in the rat (incisors-absent ia/ia, osteopetrosis op/op and toothless tl/tl) to test the hypothesis that reduced bone resorption in these mutations results in reduced tissue properties of bone material. No significant differences in elastic modulus or hardness were found between osteopetrotic mutants and their normal littermates (NLMs) in any of the three stocks. This indicates that the tissue properties of bone material are not changed significantly in osteopetrosis, even if the mechanical strength is decreased at the macroscopic level.     

Congenitally osteosclerotic (oc/oc) mice are resistant to cure by transplantation of bone marrow or spleen cells from normal littermates

The osteosclerotic mouse is a new, lethal recessive skeletal mutation which inherits osteopetrosis as an autosomal recessive. Affected mice are hypocalcemic and also have rickets. Osteoclasts are small and numerous with little evidence of function. This report examines the effects of bone marrow and spleen cell transplantation from normal littermates on survival and the radiographic appearance of the skeleton in 29 mutants. This procedure did not significantly prolong life or effect radiographic changes in the skeleton of most (27) recipients. However, two treated mutants lived for almost a year with radiographically normal skeletons. These variable responses to stem cell transplantation in an osteopetrotic mutant with rickets deserve further study.     

Organ culture of osteopetrotic (ia) rat bone: evidence that the defect is cellular.

Calvarial bone from osteopetrotic (ia) rats and normal littermates has been cultured in a chemically defined medium supplemented with homologous serum to test for the presence of inhibitors or the absence of promoters of bone resorption in mutant serum. In addition, the response of mutant and normal bone to parathyroid extract and hydrocortisone was tested in vitro. The results indicate that mutant and normal serum do not differ with respect to their ability to support bone resorption and that ia bone responds to hydrocortisone but not parathyroid extract in organ culture. These data indicate that the skeletal defect in ia rats is not humoral but cellular.     

Transformations of osteoclast phenotype in ia rats cured of congenital osteopetrosis

The reduced bone resorption characteristic of osteopetrosis is accompanied in the incisors-absent (ia) rat mutation by a significant increase in osteoclasts of inactive (mutant) phenotype. Restoration of bone resorption in ia rats by transfer of spleen cells from normal littermates is preceded by a transformation of osteoclasts from mutant to normal phenotype. In this investigation the proportions of osteoclasts of normal phenotype have been determined by light microscopy in untreated ia and normal rats and in ia rats treated with various cell populations from normal rats. Significant increases in numbers of osteoclasts of normal phenotype were seen in the mutant skeleton soon after cell treatments that eventually restored bone resorption and cured the disease. No changes in osteoclast phenotype were seen after cell transfers that did not cure the disease. These data establish transformation of osteoclast phenotype as an early event in the recovery from osteopetrosis and suggest that determination of osteoclast phenotype is a reliable predictor of the success of normal cell populations to restore bone resorption in this mutation.     

Prostaglandin D2 formation and characterization of its synthetases in various tissues of adult rats

When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 degrees C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140-340); and weakly in other tissues (less than 100). Addition of exogenous arachidonic acid (1 mM) accelerated the formation of prostaglandin D2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600-5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380-1000); and other tissues (80-310). The activity of prostaglandin D synthetase (prostaglandin-H2 D-isomerase) was detected in 100,000g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8-9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase (Y. Urade, N. Fujimoto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410-12415). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0-57.1) was glutathione-requiring prostaglandin D synthetase (Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820-3825). The activity in the kidney and testis (3.7-4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6-5.1) was due to both the glutathione-requiring synthetase and the transferase.     

Pluripotent hemopoietic stem cells give rise to osteoclasts in vitro: effects of rGM-CSF

Studies involving bone marrow transplantation of osteopetrotic rodents have provided evidence for the lineage of the ostcoclast. Recent investigations have demonstrated that pluripotent hemopoietic stem cells (PHSC) isolated from the bone marrow of normal animals cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia osteopetrotic rats. A criticism of these findings is that the microenvironment of the osteopetrotic bone and the bone marrow compartment may be unique in its ability to induce the differentiation of these stem cells into osteoclasts. To test this hypothesis, PHSC were co-cultured with fetal metatarsal bones from normal animals. PHSC were isolated from normal bone marrow using FITC-labelled monoclonal antibodies directed against rat Thy 1.1 and fluorescence-activated cell sorting. The PHSC or whole mononuclear bone marrow were co-cultured with 20-day fetal rat metatarsal rudiments. In some cultures, recombinant mouse granulocyte-macrophage colony-stimulating factor (rGM-CSF) (250 U per culture) was added in addition to the PHSC. After 7 days the fetal bones were prepared for light and electron microscopy and the number of osteoclasts generated in vitro was determined. The PHSC isolate generated as many osteoclasts as the whole mononuclear bone marrow. The addition of rGM-CSF did not enhance the generation of osteoclasts in either control bones or in bones cultured with PHSC. These results are equivalent to those reported in the osteopetrotic transplant system.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Pathological effects of the radiation protector WR-151327 in mice

The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphosphorothioic acid (WR-151327), were studied in unirradiated B6CF1 male mice. Fifty mice were injected intraperitoneally with 540 mg/kg WR-151327, and groups of five mice were sacrificed at 14-day intervals up to and including 140 days post-treatment. Ten mice served as sham-injected controls. A necropsy was performed and gross morphological abnormalities were noted. Tissues (brain, eyes, harderian gland, salivary glands, sternal bone marrow, thyroid, lung, thymus, esophagus, trachea, skeletal muscle, heart, liver, kidney, adrenal gland, spleen, small intestine, pancreas, and testes) were fixed in 10% formalin, embedded in paraffin, and sectioned. Slides were routinely stained with hematoxylin and eosin while Alizarin red stain was used to test specifically for the presence of calcium salts. Histopathological effects of WR-151327 were restricted to the testes, salivary gland, and pancreas. The caudal pole of the testes was observed to undergo progressive changes from coagulation necrosis to dystrophic calcification. The cells of the submandibular salivary gland showed mainly hyperchromatic nuclei while the pancreas showed enlarged islets of Langerhans.     

Cure of osteopetrosis by injection of allogenic bone marrow into "op" rats treated with cyclosporin A

The cure of osteopetrosis by allogenic bone marrow injection has been obtained in "op" mutant rat kept under cyclosporin A treatment. This immunosuppressive agent able to prevent the rejection of transplanted cells does not impair the propriety of these cells to restore the bone resorption process in this severe osteopetrotic form.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

Carbonic anhydrase II and H+-ATPase in osteoclasts of four osteopetrotic mutations in the rat

 Osteopetrosis in laboratory animals is a metabolic bone disease characterized by increased skeletal mass. It is inherited as an autosomal recessive and results from a defect in the development and/or function of osteoclasts. We studied two enzymes essential for bone resorption, carbonic anhydrase II isoenzyme (CA II) and H⁺-ATPase, in osteoclasts from four osteopetrotic mutations in the rat; namely incisors-absent (ia), osteopetrosis (op), toothless (tl), and microphthalmia (mib), to test the hypothesis that reduced bone resorption in one or more of these mutations results from defects in the synthesis or activity of one of these enzymes. CA II was present in most osteoclasts from normal, tl, op, and mib littermates and was homogeneously distributed in cytoplasm. CA II staining in ia osteoclasts was more variable and less intense than in the other mutations. H⁺-ATPase was also present in osteoclasts from normal animals and mutants and immunostaining showed clear polarization to the ruffled border region in all normal rats and mutants except ia, which showed diffuse distribution of staining in the cytoplasm. H⁺-ATPase activity (proton transport) in a related tissue, kidney, was normal in tl and ia rats but increased in op and mib rats compared to their normal littermates. These results suggest that the osteoclasts in osteopetrotic rat mutations are not abnormal with respect to the distribution of CA II and H⁺-ATPase and that the function of these enzymes in the skeleton, while likely normal, needs to be tested directly in bone. Accepted: 25 September 1998     

Osteoclast activity modulates B-cell development in the bone marrow

B-cell development is dependent on the interactions between B-cell precursors and bone marrow stromal cells, but the role of osteoclasts (OCLs) in this process remains unknown. B lymphocytopenia is a characteristic of osteopetrosis, suggesting a modulation of B lymphopoiesis by OCL activity. To address this question, we first rescued OCL function in osteopetrotic oc/oc mice by dendritic cell transfer, leading to a restoration of both bone phenotype and B-cell development. To further explore the link between OCL activity and B lymphopoiesis, we induced osteopetrosis in normal mice by injections of zoledronic acid (ZA), an inhibitor of bone resorption. B-cell number decreased specifically in the bone marrow of ZA-treated mice. ZA did not directly affect B-cell differentiation, proliferation and apoptosis, but induced a decrease in the expression of CXCL12 and IL-7 by stromal cells, associated with reduced osteoblastic engagement. Equivalent low osteoblastic engagement in oc/oc mice confirmed that it resulted from the reduced OCL activity rather than from a direct effect of ZA on osteoblasts. These dramatic alterations of the bone microenvironment were disadvantageous for B lymphopoiesis, leading to retention of B-cell progenitors outside of their bone marrow niches in the ZA-induced osteopetrotic model. Altogether, our data revealed that OCLs modulate B-cell development in the bone marrow by controlling the bone microenvironment and the fate of osteoblasts. They provide novel basis for the regulation of the retention of B cells in their niche by OCL activity.     

Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.

The ontogeny of hemopoietic cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in rats and mice. During fetal life, TdT-positive cells were first detected in the thymus, where they appeared on or about day 17 of gestation. TdT-positive cells were not found in fetal liver, spleen, or bone marrow, but appeared in bone marrow and spleen on the day after birth. In the rat, peak levels of TdT-positive cells were attained at 3 to 4 weeks of age in thymus, bone marrow, and spleen, accounting for 67, 3.9, and 2.3% of nucleated cells, respectively. The percentages of TdT-positive cells in thymus and bone marrow decreased gradually thereafter, whereas, TdT-positive cells in spleen were no longer detectable by 7 weeks of age. Normal percentages of TdT-positive cells were found in bone marrow and spleen from neonatally thymectomized rats and congenitally athymic (nu/nu) mice. Dexamethasone treatment resulted in a marked decrease in TdT-positive cells. The results are discussed with respect to the putative role of TdT-positive hemopoietic cells as thymocyte progenitors.