Willy Hofstetter

Characterization and cloning of the E11 antigen, a marker expressed by Rat Osteoblasts and Osteocytes

A new marker for cells of the osteoblastic lineage was identified by raising monoclonal antibodies against an immortalized rat osteoblastic cell line. Among the different antibodies one was selected which, on tissue sections, strongly reacts with osteoblasts, preosteocytes, and osteocytes. This antibody, designated E11, recognizes an antigen localized at the cell surface. The cDNA encoding the E11 antigen was cloned from a cDNA library prepared from ROS 17/2.8 cells, using a eukaryotic expression system. The E11 cDNA sequence revealed homology with the murine OTS-8/gp38 sequence. In situ hybridization confirmed that E11 mRNA expression in bone is restricted to osteoblasts and osteocytes. The tissue specificity of the E11 expression was studied by immunohistochemistry and Northern blot analysis. Apart from bone, E11-positive cells were also found in lung: namely, the alveolar cells of type I. Epithelial cells of the choroid plexus and endothelial cells of lymphatic vessels were also labeled with mAb E11. These results were confirmed by Northern blot, as the 1.8 kb E11 mRNA transcript was detected in bone and also in lung, brain, and skin. In conclusion, we describe a novel osteoblastic product which is expressed by mature osteoblasts and newly formed osteocytes.

Early osteoarthritic changes of human femoral head cartilage subsequent to femoro-acetabular impingement☆

OBJECTIVE To use the surgical samples of patients with femoro-acetabular impingement due to a nonspherical head to analyze tissue morphology and early cartilage changes in a mechanical model of hip osteoarthritis (OA). DESIGN An aberrant nonspherical shape of the femoral head has been assumed to cause an abutment conflict (impingement mechanism) of the hip with subsequent cartilage lesions of the acetabular rim and surface alterations of the nonspherical portion of the head. In this study, 22 samples of the nonspherical portions of the head have been obtained during hip surgery from young adults (mean 30.4 years, range 19-45 years) with an impingement conflict. The samples were first compared with tissue from the same area obtained from six age-matched deceased persons (control group) with normal hip morphology and second with cartilage from 14 older patients with advanced OA. All samples were characterized histologically and hyaline cartilage was graded according to the Mankin criteria. They were further subjected to examination on a molecular basis by immunohistology for cartilage oligomeric matrix protein (COMP), tenascin-C and a collagenase cleavage product (COL2-3/4C(long)) and by in situ hybridization for collagen type I and collagen type II. RESULTS All samples from the patient group revealed hyaline cartilage with degenerative signs. According to the Mankin criteria, the cartilage alterations were significantly different when compared with the control group (p=0.007) but were less distinct when compared with cartilage from patients with advanced OA (p=0.014). Positive staining and distribution pattern for COMP, tenascin-C and COL2-3/4C(long) showed similarities between the samples from the impingement group and osteoarthritic cartilage but they were distinctly different when compared with healthy cartilage. Levels of collagen I and II transcripts were upregulated in 6 and 10, respectively, of the 14 samples with OA and in 9 and 12, respectively, of the 22 samples from the impingement group. None of the samples from the control group showed upregulation of Collagen I and II mRNA. CONCLUSIONS The aberrant nonspherical portion of the femoral head in young patients with an impingement conflict consists of hyaline cartilage which shows clear degenerative signs similar to the findings in osteoarthritic cartilage. The tissue alterations are distinctly different when compared with a control group, which substantiates an impingement conflict as an early mechanism for degeneration at the hip joint periphery.

Prevalence of cam-type deformity on hip magnetic resonance imaging in young males: a cross-sectional study.

To determine the prevalence of cam‐type deformities on hip magnetic resonance imaging (MRI) in young males.

Expression of matrix proteins during the development of mineralized tissues

The specific properties of mineralized tissues are defined by the composition of the fraction of the noncollagenous matrix proteins. Because these proteins play a pivotal role in the processes of cell differentiation and activation and of mineralization, their temporal and spatial expression is tightly regulated. Within this study, the expression of the enamel protein amelogenin and of the bone matrix proteins osteopontin, bone sialoprotein, osteocalcin, and osteonectin was investigated by in situ hybridization. Two models that allow observation of the formation of mineralized tissues were chosen. The development of bone and cartilage was observed on murine metatarsals from 15-day-old embryos up to 1-day-old mice. This time covers the periods of initial bone formation as well as onset of resorption of mineralized cartilage and bone. To study gene expression in the mineralized tissues of the dental organ, enamel, dentin, and cementum, developing molars ranging in age from 16-day-old embryos to 14 days after delivery were chosen. Within this time frame, the molars develop from an immature state to the differentiated organ which erupts through the mandibular bone. In the developing metatarsals, osteopontin and bone sialoprotein mRNAs were detected in osteoblasts and hypertrophic chondrocytes at the onset of mineralization. In the tooth organ, only cementoblasts expressed transcripts encoding the two proteins; odontoblasts and ameloblasts did not express these genes. Osteonectin was expressed by osteoblasts and hypertrophic chondrocytes as well, whereas in the molars it was produced exclusively by odontoblasts. Osteocalcin was expressed specifically by osteoblasts in the developing metatarsals. In tooth, osteocalcin transcripts were detected in odontoblasts. Finally, amelogenin was a specific product of ameloblasts. Thus, a sequential and cell type-restricted expression of matrix proteins takes place during the development of the mineralized tissues. The expression patterns of the transcripts encoding the bone matrix proteins suggest different biological roles depending on the time and site of expression.

In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development

In vitro observations suggest that inorganic phosphate (Pi) transport plays an important functional role in osteogenic cells and in their matrix vesicles for the initiation of matrix calcification. Recent studies have shown that the type III sodium-dependent Pi transporters, Glvr-1 and Glvr-2, are expressed in human osteoblast-like cells and have suggested a potential role for type III transporters in regulated Pi handling in osteogenic cells. To address the relevance of these findings in the context of bone formation in vivo and, in particular, in relation to matrix calcification, we investigated expression of the Glvr-1 transporter by in situ hybridization in developing embryonic murine metatarsals, using human Glvr-1 cDNA as a probe. In this model of endochondral ossification, expression of transcripts encoding Glvr-1 could be detected from day 17 of embryonic development. A hybridization signal for Glvr-1 was specifically observed in a subset of hypertrophic chondrocytes and could not be detected in osteoblasts. The expression of Glvr-1 mRNA was compared with that of transcripts encoding extracellular matrix proteins. Glvr-1 mRNA expression was confined to a population of early hypertrophic chondrocytes expressing type X collagen and to slightly more mature cells that express transcripts encoding osteopontin but lack type X collagen mRNA. No Glvr-1 transcripts were detected in fully differentiated hypertrophic chondrocytes. This pattern of Glvr-1 mRNA expression was maintained throughout embryonic development until after birth. In conclusion, the Glvr-1 phosphate transporter is selectively expressed in a subset of hypertrophic chondrocytes during endochondral bone formation, in a region where matrix mineralization proceeds. This observation represents the first in vivo evidence consistent with a potential role for this phosphate transporter in matrix calcification.

Association between cam‐type deformities and magnetic resonance imaging–detected structural hip damage: A cross‐sectional study in young men

OBJECTIVE Femoroacetabular impingement may be a risk factor for hip osteoarthritis in men. An underlying hip deformity of the cam type is common in asymptomatic men with nondysplastic hips. This study was undertaken to examine whether hip deformities of the cam type are associated with signs of hip abnormality, including labral lesions and articular cartilage damage, detectable on magnetic resonance imaging (MRI). METHODS In this cross-sectional, population-based study in asymptomatic young men, 1,080 subjects underwent clinical examination and completed a self-report questionnaire. Of these subjects, 244 asymptomatic men with a mean age of 19.9 years underwent MRI. All MRIs were read for cam-type deformities, labral lesions, cartilage thickness, and impingement pits. The relationship between cam-type deformities and signs of joint damage were examined using logistic regression models adjusted for age and body mass index. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined. RESULTS Sixty-seven definite cam-type deformities were detected. These deformities were associated with labral lesions (adjusted OR 2.77 [95% CI 1.31, 5.87]), impingement pits (adjusted OR 2.9 [95% CI 1.43, 5.93]), and labral deformities (adjusted OR 2.45 [95% CI 1.06, 5.66]). The adjusted mean difference in combined anterosuperior femoral and acetabular cartilage thickness was -0.19 mm (95% CI -0.41, 0.02) lower in those with cam-type deformities compared to those without. CONCLUSION Our findings indicate that the presence of a cam-type deformity is associated with MRI-detected hip damage in asymptomatic young men.

Identification and characterization of the insulin-like growth factor I receptor in mature rabbit osteoclasts

In this study, the insulin‐like growth factor I (IGF‐I) receptor was identified in rabbit osteoclasts at mRNA and protein levels by in situ hybridization and autoradiography, respectively. Using highly purified mature osteoclasts, the IGF‐I receptor was characterized on the molecular level according to its size and its affinity and number per osteoclast by isolation of the receptor–ligand complex and by binding studies, respectively, and on the cellular level according to the response of mature osteoclasts to IGF‐I stimulation. In situ hybridization and autoradiography experiments showed that osteoclasts express IGF‐I receptor mRNA and IGF‐I binding sites. Chemical cross‐linking of125I‐IGF‐I bound to the purified mature osteoclasts and subsequent sodium dodecyl sulfide‐polyacrylamide gel electrophoresis revealed the specific binding of125I‐IGF‐I in complexes with molecular masses of 130 and 230 kD consistent with binding to the IGF‐I receptor. In competition experiments,125I‐IGF‐I binding to mature osteoclasts was dose‐dependently reduced by unlabeled IGF‐I in the picomolar range, whereas 20 nM insulin did not reduce the binding of125I‐IGF‐I binding. The calculated receptor number was 6000 per osteoclast, and the Kd was 0.10 nM. Searching for a role of the IGF‐I receptor in mature osteoclasts, we found no significant influence of IGF‐I on the levels of the proform of matrix metaloproteinase 9 and tartrate‐resistant acid phosphatase. However, the induction of nuclear fragmentation in serum‐depleted cultures of purified mature osteoclasts was dose‐dependently inhibited by IGF‐I in the picomolar range, but not by 1 nM insulin. These data show that functionally active IGF‐I receptor is present in mature osteoclasts.

TREM-1 Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1−/− mice are viable, fertile and show no altered hematopoietic compartment. In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Trem1−/− mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1−/− mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1−/− mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1−/− mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1+/+ controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Detection of transcripts and binding sites for colony-stimulating factor-1 during bone development

Colony-stimulating factor-1 (CSF-1), originally characterized as the growth factor for the cells of the mononuclear phagocytic system, has been shown to be essential for osteoclast formation. The aim of the present study was twofold: (i) to investigate the expression of transcripts encoding CSF-1; and (ii) to detect binding sites for CSF-1 during bone development. As a model, metatarsal rudiments from embryonic mice of different ages were used, an in vivo system allowing one to follow osteoclast formation. In 16-day-old embryos, proliferating osteoclast precursors are located on the outer surface of the rudiments. They differentiate subsequently to post-mitotic precursors. At 18 days, the precursors fuse and the mature osteoclasts invade the mineralized cartilage of the rudiments to excavate the future bone marrow cavity. Within this study, in situ hybridization on sections of whole paws from 17-day-old embryos revealed CSF-1 transcripts to be present in cells lining the outside of the midregion of the metatarsals. One day later, cells containing CSF-1 mRNA were found within the mineralized cartilage. The levels of transcripts encoding CSF-1 were further increased in the bone rudiments of newborn animals. Binding sites for CSF-1 on cells in close proximity of the metatarsals were detected at embryonic age 17 days, but not before. At this stage, cells binding CSF-1 were located on the periosteum of the midregion of the metatarsal rudiment. At 18 days, cells expressing high levels of CSF-1 binding sites had invaded the mineralized cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophage Colony Stimulating Factor Down-Regulates MCSF-Receptor Expression and Entry of Progenitors into the Osteoclast Lineage

Macrophage colony‐stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF‐receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose‐dependently decreased125I‐MCSF binding (by 65 ± 5.0% at 3500 and 87 ± 16.7% at 7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8‐ and 6.3‐fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF‐induced change in MCSF receptor‐bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2–4 dose‐dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 ± 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 ± 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF‐receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.