Brain Abscess in a Patient with Osteopetrosis: A Rare Complication

Brain abscess formation is extremely rare in patients with osteopetrosis. Herein, we report a case of viridans streptococci brain abscess in an immunocompromised child diagnosed with osteopetrosis. The patient presented with a sudden change in mental status and convulsions. Radiological evaluation revealed a temporal lobe brain abscess, and intravenous antibiotherapy was started immediately. The patient underwent abscess drainage, and laboratory investigation of pus material revealed viridans streptococci.

A monoclonal antibody ameliorates local inflammation and osteoporosis by targeting TNF-α and RANKL.

This study aimed to generate a monoclonal antibody (mAb) targeting both tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL) and to evaluate the therapeutic effects of this antibody on acute inflammation and osteoporosis. We used hybridoma techniques to generate potential mAbs and enzyme-linked immunosorbent assay (ELISA) to determine their specificity. Crystal violet staining was performed to measure the effective dose of the candidate mAbs. The neutralizing effect of the mAbs was evaluated by TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays. We further assessed the therapeutic effect of the mAbs in BALB/c mice with carrageenan-induced acute inflammation and ovariectomy-induced osteoporosis. We successfully generated an IgG1 isotype mAb that recognizes human TNF-α and RANKL, which we named 8G12. The 50% effective dose of 8G12 was approximately 1µg/mL. L929 cells treated with 8G12 exhibited decreased levels of apoptosis (20.04% compared to 63.28% in the positive controls). In addition, treatment with 8G12 inhibited osteoclastogenesis in a dose-dependent manner in vitro. Carrageenan-induced paw edema was significantly reduced in the 8G12-treated mice compared to the positive controls. Treatment with 8G12 also reduced the number of infiltrating leukocytes by more than 50%. The 8G12 treatment not only prevented bone loss but also increased the number, thickness and volume of trabeculae and reduced trabecular separation in ovariectomized mice. Our data suggest that the 8G12 effectively neutralizes the bioactivity of TNF-α and RANKL, ameliorating osteoporosis and inflammation. We therefore propose that 8G12 could be a candidate for generating therapeutic antibodies for treating inflammatory bone diseases.

RANKL cytokine: from pioneer of the osteoimmunology era to cure for a rare disease.

Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.

The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation.

Jdp2 is an AP-1 family transcription factor that regulates the epigenetic status of histones. Previous in vitro studies revealed that Jdp2 is involved in osteoclastogenesis. However, the roles of Jdp2 in vivo and its pleiotropic functions are largely unknown. Here we generated Jdp2(-/-) mice and discovered its crucial roles not only in bone metabolism but also in differentiation of neutrophils. Jdp2(-/-) mice exhibited osteopetrosis resulting from impaired osteoclastogenesis. Jdp2(-/-) neutrophils were morphologically normal but had impaired surface expression of Ly6G, bactericidal function, and apoptosis. We also found that ATF3 was an inhibitor of neutrophil differentiation and that Jdp2 directly suppresses its expression via inhibition of histone acetylation. Strikingly, Jdp2(-/-) mice were highly susceptible to Staphylococcus aureus and Candida albicans infection. Thus, Jdp2 plays pivotal roles in in vivo bone homeostasis and host defense by regulating osteoclast and neutrophil differentiation.

The macrophage system in the intestinal muscularis externa during inflammation: an immunohistochemical and quantitative study of osteopetrotic mice.

Intestinal inflammation results in disturbed intestinal motility in humans as well as in animal models. This altered function of smooth muscle cells and/or the enteric nervous system may be caused by activation of macrophages in muscularis externa and a thereby following release of cytokines and chemokines that causes influx of mononuclear cells and neutrophilic granulocytes. We subjected osteopetrotic (op/op) mice that lack certain macrophage subtypes, e.g. macrophages in the muscularis externa and +/+ mice to LPS to induce inflammatory cell influx. The densities of F4/80+, MHCII+, and myeloperoxidase+ cells were quantified using stereological sampling. In +/+ mice we found that MHCII+ cells outnumber F4/80+ cells and that LPS injection increased the density of MHCII+ cells temporarily but not that of F4/80+ cells. This indicates that an upregulation of MHCII antigen takes place and that two or more macrophage subtypes with comparable morphologies exist. Osteopetrotic mice lacked MHCII+, CD169+, and F4/80+ cells after either treatment, which indicate that these cells are CSF-1-dependent. LPS induced VCAM-1 activation of the vessels, modest influx of granulocytes, as well as an iNOS-activation in a cell type different from macrophages in both +/+ and op/op mice.

Resistance artery remodeling in deoxycorticosterone acetate-salt hypertension is dependent on vascular inflammation: evidence from m-CSF-deficient mice.

Deoxycorticosterone acetate (DOCA)-salt hypertension has an important endothelin-1 (ET-1)-dependent component. ET-1-induced vascular damage may be mediated in part by oxidative stress and vascular inflammation. Homozygous osteopetrotic (Op/Op) mice, deficient in macrophage colony-stimulating factor (m-CSF), exhibit reduced inflammation. We investigated in osteopetrotic (Op/Op) mice the effects of DOCA-salt hypertension on vascular structure, function, and oxidative stress, the latter as manifested by reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase activity. Mice were implanted with DOCA (200 mg/mouse, under 5% isofluorane anesthesia) and given saline for 14 days. Systolic blood pressure (mmHg) was significantly increased (146 +/- 2 and 138 +/- 1; P < 0.001 vs. basal 115 +/- 3 and 115 +/- 3, respectively) by DOCA-salt in wild-type (+/+) and heterozygous (Op/+) mice, but not in Op/Op mice (130 +/- 1 vs. basal 125 +/- 3). Norepinephrine contractile response was significantly enhanced, while acetylcholine endothelium-dependent vasodilation was significantly impaired in DOCA-salt-treated +/+ and Op/+ mice compared with control mice. No changes in norepinephrine-induced contraction and acetylcholine-induced relaxation were observed in DOCA-salt Op/Op mice. DOCA-salt +/+ and Op/+ mice had significantly increased mesenteric resistance artery media-to-lumen ratio and media cross-sectional area, neither of which were altered in Op/Op mice. Basal vascular superoxide production and NAD(P)H oxidase activity, vascular cell adhesion molecule-1 expression, and macrophage infiltration were significantly increased only in DOCA-salt +/+ mice. Thus m-CSF-deficient mice developed less endothelial dysfunction, vascular remodeling, and oxidative stress induced by DOCA-salt than +/+ and Op/+ mice, suggesting that inflammation may play a role in DOCA-salt hypertension, a model that results in part from effects of ET-1, which has proinflammatory actions.

Osteopetroses and immunodeficiencies in humans.

PURPOSE OF REVIEW: This review focuses on human and murine pathologies involving both osteoclasts and immune cells. These diseases have been relevant to the discovery of novel interactions and pathways shared between these two types of cells. RECENT FINDINGS: Interactions between immune cells and osteoclasts were originally shown in murine models by gene targeting of molecules involved in the early steps of osteoclast differentiation, since receptor activator of nuclear factor kappa-B ligand (RANKL), RANK and TNFR-associated factor 6 knockout mice bore abnormalities of both bone resorption and immune system. Subsequently, osteoclast stimulation by RANKL secreted by lymphocytes in autoimmune diseases, such as rheumatoid arthritis, was found. More recently, the identification of immunoreceptor tyrosine-based activation motif receptors and adaptors important for both dendritic cells and osteoclast function has established a link between innate and adaptive immunity and bone. Finally, osteoclasts are also important for hematopoietic stem-cell mobilization, providing a further level of regulation of lymphoid cells. SUMMARY: These findings open up a new field of research, osteoimmunology, which will unravel previously unsuspected links between bone remodelling and the immune response.

Inflammation and atherosclerosis: novel insights into plaque formation and destabilization.

BACKGROUND AND PURPOSE: The simplistic view of atherosclerosis as a disorder of pathological lipid deposition has been redefined by the more complex concept of an ongoing inflammatory response. SUMMARY OF REVIEW: Apolipoprotein E and low-density lipoprotein (LDL)-receptor-deficient mice develop accelerated atherosclerosis allowing in-depth pathophysiological investigations. Atherosclerotic plaques in these mice contain large numbers of T cells and macrophages. Crossbreeding apolipoprotein E-deficient mice with T-cell-deficient mice and mice with impaired macrophage function (osteopetrotic op/op mice) disclosed the important impact of immune cells on atherosclerotic lesion development. In contrast to the detrimental role of T cells and macrophages, B cells appear to be atheroprotective. These basic experimental findings have partly been confirmed in studies of the human carotid artery system. Inflammation is not only instrumental in the development of human atheromatous plaques, but, importantly, plays a crucial role in the destabilization of internal carotid artery plaques, thus converting chronic atherosclerosis into an acute thrombo-embolic disorder. Humoral factors involved in internal carotid artery destabilization include cytokines, cyclooxygenase-2, matrix metalloproteinases, and tissue factor. Antibodies to oxidized LDL can reflect disease activity on one hand, but can also confer atheroprotection. Novel MRI techniques may aid in the in vivo assessment of acute plaque inflammation in humans. CONCLUSIONS: The impact of inflammation on the development of atherosclerotic plaques and their destabilization opens new avenues for treatment. The effects of statins, acetylsalicyclic acid and angiotensin-converting enzyme inhibitors on stroke prevention may partly be attributable to their profound anti-inflammatory actions. Vaccination against modified LDL and heat shock proteins halt plaque progression in experimental atherosclerosis. Their potential for prevention of human atherosclerosis is currently under investigation.

Partial rescue of B cells in microphthalmic osteopetrotic marrow by loss of response to type I IFNs.

The microphthalmic (mi) mouse exhibits deficiencies in the development of osteoclasts, melanocytes, mast cells and marrow B cells. Previously, we demonstrated that the marrow of such mice over-express receptor activator of nuclear factor kappaB (RANK) ligand (RANKL). RANKL has been shown to induce the production of IFN-beta, a type I IFN. Additionally, maturing B cells have been shown to undergo apoptosis in response to type I IFNs including IFN-beta during differentiation. We hypothesized that the loss of B cells in the marrow of mi mice was due to the over-expression of IFN-beta as a result of heightened RANK-RANKL signaling. Creating a mouse with the mi genotype that was non-responsive to IFN-beta (lacking the type I IFNR) allowed us to test this hypothesis. These mice demonstrated an elevated number of marrow B cells and marrow precursor cells compared with mi animals possessing the type I IFNR. Intriguingly, type I IFNR-deficient wild-type animals also demonstrated an increased number of precursor cells in the marrow, but not an expansion of B220-positive pre-B cells, compared with wild type, suggesting that modulation of type I IFN responses directly controls the development of marrow constituents.

Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-infused macrophage colony-stimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury.

OBJECTIVE: Angiotensin (Ang) II-induced vascular damage may be partially mediated by reactive oxygen species generation and inflammation. Homozygous osteopetrotic mice (Op/Op), deficient in macrophage colony-stimulating factor (m-CSF), exhibit reduced inflammation. We therefore investigated Ang II effects on vascular structure, function, and oxidant stress generation in this model. METHODS AND RESULTS: Adult Op/Op, heterozygous (Op/+), and wild type (+/+) mice underwent 14-day Ang II (1000 ng/kg per minute) or saline infusion. Blood pressure (BP) was assessed by radiotelemetry, mesenteric resistance artery vascular reactivity was studied on a pressurized myograph, and vascular superoxide and NAD(P)H oxidase activity by lucigenin chemiluminescence. Ang II increased BP in Op/+ and +/+ mice but not in Op/Op. Ang II-treated Op/+ and +/+ mice showed reduced acetylcholine-mediated relaxation (maximal relaxation, respectively, 64% and 67% versus 84% and 93% in respective controls; P<0.05), which was unaffected by L-NAME. Ang II-infused Op/Op mice arteries showed significantly less endothelial dysfunction than vehicle-infused counterparts (maximal relaxation 87% versus 96% in shams). Resistance arteries from Ang II-infused +/+ and Op/+ mice had significantly increased media-to-lumen ratio and media thickness, neither of which was altered in Op/Op mice compared with untreated littermates. Vascular media cross-sectional area, NAD(P)H oxidase activity and expression, and vascular cell adhesion molecule (VCAM)-1 expression were significantly increased by Ang II only in +/+ mice (P<0.05). CONCLUSIONS: m-CSF-deficient mice (Op/Op) developed less endothelial dysfunction, vascular remodeling, and oxidative stress induced by Ang II than +/+ littermates, suggesting a critical role of m-CSF and proinflammatory mediators in Ang II-induced vascular injury.

Characterization of a novel bipotent hematopoietic progenitor population in normal and osteopetrotic mice.

UNLABELLED: Several reports indicate that osteoclasts and B-lymphocytes share a common progenitor. This study focuses on the characterization of this bipotent progenitor from the bone marrow of the osteopetrotic oc/oc mouse, where the bipotent progenitor population is amplified, and of normal mice. INTRODUCTION: Osteoclasts have a myelomonocytic origin, but they can also arise in vitro from pro-B-cells, suggesting that a subset of normal pro-B-cells is uncommitted and may reorient into the myeloid lineage representing a B-lymphoid/osteoclastic progenitor. The aim of this study was to characterize this progenitor population. MATERIALS AND METHODS: The osteopetrotic oc/oc mouse was used as a choice model because it displays an increased number of both osteoclasts and pro-B-cells in the bone marrow. Our results have been confirmed in normal littermates. Bone marrow cells from these animals were analyzed by flow cytometry. After sorting, the cells were cultured under different conditions to assess their differentiation capacity. RESULTS: Pro-B-cells from oc/oc and normal mice include an unusual biphenotypic population expressing markers from the B-lymphoid (CD19, CD43, CD5) and the myeloid (F4/80) lineages. This population also expresses progenitor markers (CD34 and Flt3) and is uncommitted. After sorting from the oc/oc bone marrow, this population is able to differentiate in vitro into osteoclast-like cells in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), into dendritic-like cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and TNFalpha, and into immature B-cells when seeded onto ST2 cells in the presence of IL-7. CONCLUSION: Our results show the existence of a novel bipotent biphenotypic hematopoietic progenitor population present in the bone marrow that has retained the capacity to differentiate into myeloid and B-lymphoid cells.

Periwound dopaminergic sprouting is dependent on numbers of wound macrophages.

Injury to many regions of the central nervous system, including the striatum, results in a periwound or 'abortive' sprouting response. In order to directly evaluate whether macrophages play an important role in stimulating periwound sprouting, osteopetrotic (op/op) mice, which when young are deficient in a variety of macrophage subtypes, were given striatal wounds and the degree of dopaminergic sprouting subsequently assessed. Two weeks postinjury, significantly fewer wound macrophages were present in the striata of op/op mice compared with controls (144 +/- 30.1 in op/op mice vs. 416.6 +/- 82.3 in controls, P < 0.005, analysis performed on a section transecting the middle of the wound). Dopamine transporter immunohistochemistry revealed a marked decrease in the intensity of periwound sprouting in the op/op group of animals. Quantification of this effect using [H3]-mazindol autoradiography confirmed that periwound sprouting was reduced significantly in the op/op mice compared with controls (71.4 +/- 21.7 fmol/mg protein in op/op mice vs. 210.7 +/- 27.1 fmol/mg protein in controls, P < 0.0005). In the two groups of animals the magnitude of the sprouting response in individuals was closely correlated with the number of wound macrophages (R = 0.83, R2 = 0.69). Our findings provide strong support for the crucial involvement of macrophages in inducing dopaminergic sprouting after striatal injury.

Absence of CSF-1-dependent macrophages does not improve function of transplanted islets of Langerhans.

A role of macrophage-mediated inflammatory events in early islet graft loss is increasingly acknowledged. Osteopetrotic mice (op/op) have a complete absence of CSF-1, and thus of most tissue macrophages. We have investigated whether the absence of CSF-1-dependent macrophages in the graft itself or at the transplant site could decrease the delay to function of a syngeneic marginal islet mass. Islets transplanted into op/op or control recipients reversed diabetes in 59 days vs. 10 days (p = 0.28, NS). Islets isolated from op/op or control mice reversed diabetes in 11 days vs. 10 days. IL-1 and TNF-alpha release by cultured islets was markedly decreased for op/op islets compared with control islets (IL-1: 0 vs. 4.2 pg/ml, p = 0.07; TNF-alpha: 67 vs. 311 pg/ml, p = 0.002). In contrast, IL-6 release by op/op islets was significantly increased (11.1 vs. 4.3 ng/ml, p = 0.006). CSF-1-dependent tissue macrophages may not be critical in the inflammatory insult to islet transplants. Alternate patterns of intraislet release of deleterious proinflammatory cytokines may exist.

Lipopolysaccharide-induced cytokine and receptor expression and neutrophil infiltration in the liver of osteopetrosis (op/op) mutant mice.

BACKGROUND/AIMS: Mice homozygous for the osteopetrosis (op) mutation are genetically deficient in macrophage colony-stimulating factor (M-CSF/CSF-1) and are characterized by defective differentiation and function of macrophages. The aim of this study is to assess the contribution of M-CSF to lipopolysaccharide (LPS)-induced cytokine expression and neutrophil infiltration in the liver. METHODS: We investigated the effects of LPS administration in M-CSF-deficient op/op mutant mice. The expression of cytokines and receptors in the liver was studied by immunohistochemistry and RT-PCR. Neutrophil infiltration in the liver was also examined. RESULTS: After LPS administration, cytokine production and expression of LPS receptors, such as CD14 and scavenger receptor class A (MSR-A), were induced at lower levels in op/op mice than those in littermate mice. Neutrophil infiltration in the liver of op/op mice did not differ significantly from that of littermate mice. Anti-IL-8 receptor homologue and anti-C5a receptor antibody reduced the number of infiltrating neutrophils. CONCLUSIONS: These findings indicate that deficient macrophage activation following LPS injection in op/op mice is associated with decreased expression of CD14 and MSR-A in the liver. Thus, M-CSF plays a critical role in LPS-induced macrophage activation but does not exert a dominant role in neutrophil infiltration in the liver.

Insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) immunoreactivity in normal and osteopetrotic (toothless, tl/tl) rat tibia.

Insulin-like growth factor-I (IGF-I) plays a major role in regulating cell growth. This study examined the immunohistochemical distribution of IGF-I and IGF-I receptor (IGF-IR) in tibias from normal and osteopetrotic (toothless, tl/tl) rats, following treatment with colony stimulating factor-1 (CSF-1). In normal rats, immunoreactivity for IGF-I and IGF-IR was detected in cells of the articular and epiphyseal cartilage, secondary ossification centres, zones of resting and proliferating chondrocytes and bone marrow. Bone marrow cells immunoreactive for IGF-I and IGF-IR were significantly reduced in the tl/tl rat (p < 0.001) compared with normal animals. Treatment of tl/tl rats with CSF-1 increased immunoreactivity for IGF-I and IGF-IR in bone marrow cells as well as the number of TRAP positive osteoclasts. This increase was the result of recruitment of a range of hematopoietic cell types, including eosinophils, polymorphs and a substantial number of monocyte-like cells demonstrating strong immunoreactivity to IGF-I/IGF-IR. The differences in relative immunoreactivity for IGF-I/IGF-IR by bone marrow cells in untreated and CSF-1-treated tl/tl rats indicate a CSF-1-dependent recruitment of cells bearing surface IGF-IRs which may be mediated by an increase in local or systemic IGF-I.

Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice.

Osteopetrotic (op/op) mice lack functional M-CSF and have depressed levels of macrophages and osteoclasts. We prepared transgenic mice (hMRP8bcl-2) that express human Bcl-2 in monocytes. In vitro hMRP8bcl-2 monocytes do not undergo apoptosis in the absence of serum and M-CSF, while op/op and wild-type monocytes die. These Bcl-2-expressing monocytes spontaneously undergo macrophage differentiation. In vivo, the op/op hMRP8bcl-2 mice show significant replenishment of tissue macrophages. Their long bone osteopetrosis is largely reversed, and extensive medullary hematopoiesis appears in the bone marrow. We propose that M-CSF augments monocyte survival, permitting them to respond to internal and external cues for their differentiation.

Cytophilic immunoglobulin G binding on neutrophils from a child with malignant osteopetrosis who developed fatal acute respiratory distress mimicking transfusion-related acute lung injury.

A 16-month-old boy, diagnosed at age 3 months with osteopetrosis, was treated since age 6 months with rhIFN-gamma in combination with rhM-CSF. The child developed acute respiratory distress within 1 hr of a paternal platelet transfusion. Both the child and the father were blood group type O, and platelets were collected the previous day from the father. Chest X-ray revealed right pulmonary consolidation and a complete "whiteout" on the left. By 24 hr, the lungs had the appearance of adult respiratory distress syndrome (ARDS). Over the course of the next 11 days, the child remained intubated and hypotensive, and died of respiratory insufficiency 11 days later. ARDS was confirmed at autopsy. Pre- and posttransfusion patient's sera, as well as paternal serum, were tested by granulocyte agglutination and flow cytometry against granulocytes (PMN) from the patient, father, mother, and routine cell-panel donors and lymphocytes for the presence of neutrophil-specific and lymphocyte (HLA) antibodies, to rule out classical transfusion-related acute lung injury (TRALI). Both the patient's and the paternal sera were devoid of antibodies, but the patient's neutrophils demonstrated strong binding of cytophilic IgG accompanied by extremely low serum IgG and IgG1 levels. Since rhIFN-gamma is known to upregulate Fc gamma receptor type I (Fc(gamma)RI) with high affinity for IgG1, the binding of cytophilic IgG suggests that the patient's neutrophils may have been activated in vivo. The case report of another child with osteopetrosis has also been described. Although the blood specimen was not available for serological studies, this 4 1/2-year-old child treated with rhIFN-gamma and rhM-CSF also died of adult respiratory distress syndrome, with similar clinical presentations.

A defect in inducible beta-galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso-Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso-Pc efficiently activated macrophages whereas lyso-Pc-treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso-Pc-inducible beta-galactosidase of B lymphocytes. Lyso-Pc-inducible beta-galactosidase of B lymphocytes was found to be defective in mi mutant mice.