A role for cell-surface CSF-1 in osteoblast-mediated osteoclastogenesis.

CSF-1 is required for osteoblast-mediated osteoclast formation. Osteoblasts produce soluble (sCSF-1) and cell-surface forms of CSF-1 (also known as membrane-bound CSF-1, mCSF-1) but their individual contributions to osteoclastogenesis remain unclear. Using glutaraldehyde-fixed primary murine osteoblasts as a source of mCSF-1, osteoblasts from op/op mice as a source for other osteoblast-derived osteoclastogenic factors and murine bone marrow as a source of osteoclast progenitors, osteoclast-like cells (OCL) formation was observed after 7-9 days of co-culture. In contrast, no OCL formation occurred when mCSF-1 expressed by primary murine osteoblasts was blocked by CSF-1 antibody pretreatment or when op/op osteoblasts were substituted for primary murine osteoblasts in the co-culture system. Osteoclast formation was also significantly inhibited when murine primary osteoblasts were pretreated with an antisense phosphorothioate oligonucleotide against mCSF-1. Finally, mCSF-1 and sCSF-1 were synergistic in stimulating OCL formation. These data support the conclusion that mCSF-1 plays an important role in osteoblast-mediated osteoclastogenesis within the bone microenvironment.

Dual infection with Pneumocystis carinii and Pasteurella pneumotropica in B cell-deficient mice: diagnosis and therapy.

BACKGROUND AND PURPOSE: The clinical presentation, diagnosis, histopathologic findings, and elimination of dual respiratory tract infection with Pasteurella pneumotropica and Pneumocystis carinii were studied in 100 adult barrier-reared C.B17 and MRL- lpr mice homozygous for a targeted mutation of the JH region of the immunoglobulin heavy chain. METHODS: Necropsy, aerobic bacteriologic culture of hematogenous and pulmonary tissues, histochemical staining of pulmonary tissues, polymerase chain reaction analysis of pulmonary tissues and feces, and viral serologic testing were performed on 19 clinically affected mice and 8 clinically normal mice, then later on antibiotic-treated and caesarian re-derived mice. Therapeutic strategies included sequential administration of trimethoprim/ sulfamethoxazole and enrofloxacin or enrofloxacin administration and caesarian rederivation. RESULTS: Clinically affected mice had diffuse, nonsuppurative, interstitial pneumonia with superimposed pyogranulomatous lobar pneumonia that was detected microscopically. Affected lung tissue yielded pure culture of P. pneumotropica. Aged-matched, clinically normal mice of both genotypes had interstitial histiocytic pneumonia without lobar pneumonia, and P. pneumotropica was not isolated. Histochemical staining of lung tissues from normal and clinically affected mice revealed scattered cysts consistent with P. carinii, principally in the interstitium. Treatment with sulfamethoxazole/trimethoprim and enrofloxacin eliminated bacteriologic detection of P. pneumotropica, decreased mortality from 50% to 6%, and improved breeding performance. CONCLUSION: A successful antibiotic therapy and rederivation approach, incorporating enrofloxacin, cesarian section, and isolator rearing, was developed for B cell-deficient mice with opportunistic infections.

Nuclear factor-kappaB p50 is required for tumor necrosis factor-alpha-induced colony-stimulating factor-1 gene expression in osteoblasts.

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-alpha, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-kappaB site in the CSF-1 promoter is required for TNF-alpha-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-kappaB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IkappaB alpha, IkappaB gamma, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-alpha-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-alpha-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-kappaB p50 knock-out mice. Our findings support the conclusion that the NF-kappaB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

The cell-surface form of colony-stimulating factor-1 is regulated by osteotropic agents and supports formation of multinucleated osteoclast-like cells.

Colony-stimulating factor-1 (CSF-1) is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. CSF-1 is synthesized as a soluble or cell-surface protein. It is unclear, however, whether human osteoblasts express both molecular forms of CSF-1, and whether these isoforms can independently mediate osteoclastogenesis. In the present study, using a combination of quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western immunoblot analysis, we have demonstrated that human osteoblast-like cells as well as primary human osteoblasts express the cell-surface form of CSF-1 both constitutively and in response to parathyroid hormone and tumor necrosis factor. Furthermore, using an in vitro co-culture system, we have shown that cell-surface CSF-1 alone is sufficient to support osteoclast formation. These findings may be especially significant in view of evidence that direct cell-to-cell contact is critical for osteoclast formation, and suggest that differential regulation of expression of the CSF-1 isoforms may influence osteoclast function modulated by osteotropic hormones.

Colony stimulating factor-1 plays a role in osteoclast formation and function in bone resorption induced by parathyroid hormone and parathyroid hormone-related protein.

Although colony stimulating factor-1 (CSF-1) plays a key role in osteoclast recruitment, studies examining the effect of CSF-1 on mature osteoclasts indicate that it may directly inhibit bone resorption by isolated rat osteoclasts. To define further CSF-1's role in bone remodeling, we examined the effect of neutralizing antisera to CSF-1 on basal and parathyroid hormone (PTH)-induced bone resorption using two organ culture assays designed to examine the recruitment of osteoclast precursors and the activation of mature osteoclasts, respectively. We first assessed whether PTH increases CSF-1 production from bone in organ culture by examining conditioned medium from 19-day-old fetal rat long bones in a mitogenesis assay employing a CSF-1-responsive cell line, CRX-1. Conditioned medium from untreated bones induced a titratable increase in CRX-1 cell proliferation, and treatment of bones with PTH for 72 h caused a significant increase in mitogenic activity. CSF-1 antiserum caused a significant decrease in mitogenic activity in conditioned medium, indicating that bone in organ culture produces CSF-1 constitutively and in response to PTH. To examine bone-derived CSF-1's role in bone resorption, we examined the effect of neutralizing antisera to CSF-1 on basal and PTH-induced bone resorption in the fetal rat long bone assay, which reflects activation of mature osteoclasts. Anti-CSF-1 caused a significant increase in unstimulated and PTH-induced bone resorption compared with control. By contrast, in the fetal mouse metacarpal assay, which examines proliferation and recruitment of osteoclast progenitors and precursors, anti-CSF-1 caused significant inhibition of PTH related protein (PTHrP)-induced bone resorption after 3 and 6 days of incubation. Consistent with these findings, histological examination of cultured 17-day-old fetal metacarpals demonstrated that anti-CSF-1 inhibits the formation of tartrate-resistant acid phosphatase-positive osteoclasts in PTHrP-treated explants, whereas it has no effect on unstimulated bones. We conclude that bone-derived CSF-1 may have a dual role in PTH/PTHrP-induced bone resorption by enhancing the appearance of osteoclast precursors while restraining the resorptive function of mature osteoclasts.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Effects of continuous glucocorticoid infusion on bone metabolism in the rat.

The effects of continuous administration of supraphysiologic doses of dexamethasone (DEX) on bone metabolism were examined in rats. Adult, male, Sprague Dawley rats were infused with DEX at a constant rate of 16.25 microg/day for 19 days. Despite soft tissue catabolism, DEX treatment led to a significant increase in bone volume in all experiments. This was accompanied by a significant gain in femoral weight and calcium content. These findings were also observed in DEX-treated parathyroidectomized animals indicating that intact parathyroid function was not required for this effect. DEX treatment did not affect mean levels of serum calcium or phosphorus but led to significant declines in circulating levels of PTH and 1,25(OH)2D and in the urinary calcium/creatinine ratio. This latter finding was also observed in PTX animals in which 1,25(OH)2D levels did not change. Serum concentrations of osteocalcin and tartrate-resistant acid phosphatase both declined in a time-dependent manner with DEX treatment suggesting a slowing of bone turnover with the net effect favoring formation. However, histomorphometric findings were variable. Two of three experiments demonstrated a decrease in cellular parameters of formation and resorption and in one experiment, these indices increased. Mineralized surface increased with DEX treatment. We conclude that, in marked contrast to the findings in man and certain other species, DEX treatment increases bone mass in rats. This may in part relate to a relatively greater suppression of resorption vis à vis formation.

Tumor necrosis factor-alpha induces transcription of the colony-stimulating factor-1 gene in murine osteoblasts.

Tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption both in vitro and in vivo. The cellular mechanisms for this effect are not known but one pathway may be via release of osteoblast derived factors which stimulate osteoclast formation. Because colony-stimulating factor-1 (CSF-1) is essential for osteoclast progenitor proliferation, we examined the effect of TNF-alpha on osteoblast expression of CSF-1. TNF-alpha treatment of MC3T3-E1 or primary mouse osteoblasts stimulated the secretion of an activity that was mitogenic for a CSF-1 responsive cell line and was completely neutralized by antiserum to CSF-1. By Northern analysis, TNF-alpha caused a dose and time (3 to 24 h) dependent increase in CSF-1 transcript expression in MC3T3-E1 cells. mRNA stability studies using actinomycin D revealed that TNF-alpha does not affect CSF-1 mRNA half-life in MC3T3-E1 cells, while nuclear-run off analysis demonstrated that TNF-alpha increases CSF-1 gene transcription. Cycloheximide treatment of MC3T3-E1 cells up-regulated CSF-1 mRNA, and compared to either agent alone, cycloheximide and TNF-alpha in combination resulted in augmentation of CSF-1 expression. A series of studies using both agonists and inhibitors indicated that TNF-alpha-induced CSF-1 expression did not involve the arachidonic acid, PKC, or cAMP pathways. These results suggest that TNF-alpha induces CSF-1 expression in osteoblasts by a transcriptional mechanism which is largely independent of new protein synthesis and of the second messenger pathways examined.

Transactivation of the PTHrP gene in squamous carcinomas predicts the occurrence of hypercalcemia in athymic mice.

Humoral hypercalcemia of malignancy (HHM) is caused by the secretion of parathyroid hormone-related protein (PTHrP) by tumor cells, and tumors of squamous histology are the ones most commonly complicated by HHM. To determine why some squamous tumors cause HHM and others do not, we quantitated the levels of PTHrP mRNA expression and PTHrP secretion in a series of eight squamous tumor lines. As anticipated, we found that the level of PTHrP mRNA expression in individual lines correlated with their PTHrP secretion rates. However, PTHrP mRNA levels varied widely in individual lines, and only those tumor lines with the highest levels of PTHrP gene expression were able to cause hypercalcemia in athymic mice. We found that a specific segment of the PTHrP promoter could reproduce the relative pattern of PTHrP gene expression when cloned in front of a chloramphenicol acetyltransferase reporter gene and transiently transfected into these squamous lines. Deletional analysis confirmed that specific sequences within the PTHrP gene promoter appeared to be involved in the transactivation of the gene in tumor lines expressing high levels of PTHrP mRNA. These data suggest that the ability of a given squamous tumor to cause HHM is ultimately a function of its level of PTHrP gene expression, which in turn appears to be a function of the ability of specific transcription factors to transactivate PTHrP gene expression.

Defining the roles of parathyroid hormone-related protein in normal physiology.

Parathyroid hormone-related protein (PTHrP) was discovered as a result of a search for the circulating factor secreted by cancers which causes the common paraneoplastic syndrome humoral hypercalcemia of malignancy. Since the identification of the peptide in 1982 and the cloning of the cDNA in 1987, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved by prohormone convertases to yield a complex family of peptides, each of which is believed to have its own receptor. It is also clear that the PTHrP gene is expressed not only in cancers but also in the vast majority of normal tissues during adult and/or fetal life. In contrast to the situation in humoral hypercalcemia of malignancy in which PTHrP plays the role of a classical "endocrine" hormone, under normal circumstances PTHrP plays predominantly paracrine and/or autocrine roles. These apparent physiological functions are also complex and appear to include 1) regulation of smooth muscle (vascular, intestinal, uterine, bladder) tone, 2) regulation of transepithelial (renal, placental, oviduct, mammary gland) calcium transport, and 3) regulation of tissue and organ development, differentiation, and proliferation. In this review, the discovery of PTHrP, the structure of its gene and its cDNAs, and the posttranslational processing of the initial translation products are briefly reviewed. Attention is then focused on a detailed organ system-oriented review of the normal physiological functions of PTHrP.

Relative overexpression of the parathyroid hormone-related protein gene in human leiomyomas.

Uterine leiomyomas or fibroids are common among women of reproductive age, but their biology is poorly understood. The PTH-related protein (PTHrP) has been identified in a number of sites throughout the reproductive tract. We, therefore, examined whether fibroids express PTHrP mRNA and compared their level of expression with that in normal myometrium. Total RNA prepared from fibroid tissue and corresponding normal myometrium from seven patients was examined by RNase protection analysis. In all cases, fibroid and myometrial tissue expressed PTHrP, and in six of seven cases, PTHrP expression was higher in fibroids than in normal myometrium. Cultured fibroid cells from four patients also expressed higher levels of PTHrP mRNA than corresponding cultured normal myometrial cells. Tissue extracts from eight patients and conditioned medium from cultured cells from nine patients were examined for PTHrP immunoreactivity using a two-site immunoradiometric assay. In tissue extracts and conditioned medium, the mean PTHrP concentration was significantly higher in fibroids than normal myometrium. Immunohistochemical staining of fibroid and myometrial tissue was positive for PTHrP. Finally, PTHrP-(1-34) induced a dose-dependent increase in cAMP in fibroid and myometrial cells in vitro. These findings suggest that PTHrP may have an autocrine/paracrine function in regulating myometrial physiology and may play a role in regulating fibroid growth or differentiation.

Macrophage colony-stimulating factor release and receptor expression in bone cells.

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.

Infrequent shedding and transmission of herpesvirus simiae from seropositive macaques.

The epizootiologic properties of Herpesvirus simiae (B virus) were studied in singly housed macaques (Macaca mulatta and M. fascicularis) in a biomedical vivarium to determine whether commonly encountered environments and procedures such as quarantine, breeding, Caesarean section, parturition, and social stress induced virus shedding and transmission. Macaques were tested serologically and for infectious virus. Oral, conjunctival, and vaginal swab samples were obtained repeatedly. Virus excretion was not detected during a 7-week quarantine of 32 newly acquired, singly housed animals tested every other week for 6 weeks, and none of 19 seronegative animals from this group seroconverted during 7 weeks in quarantine. No virus shedding was detected in 16 seropositive animals tested weekly for 3 weeks after Caesarean section or normal parturition or in 11 seropositive animals following introduction of new males to animals rooms. One animal seroconverted after repeated breeding of seropositive animals to seronegative partners. Fifty-three singly housed offspring remained seronegative for up to 10 years, even if born to seropositive dams, and only 1 of 86 singly housed animals less than 7 years old was seropositive. These results suggest that shedding of B virus from seropositive macaques is uncommon, when subjected to common laboratory procedures or environments, and that transmission is rare in singly housed animals. These results may be useful in establishing B virus-free colonies of macaques.

Stretch-induced parathyroid hormone-related peptide gene expression in the rat uterus.

We previously observed a peak in parathyroid hormone-related peptide (PTHrP) mRNA expression in preterm rat myometrium and found that this peak was dependent on intrauterine occupancy. We explored the possibility that mechanotransduction might control PTHrP gene expression in the uterus. This was done by developing an intrauterine balloon system that allowed us to reproduce experimentally the mechanical effects of the fetal pup in utero. An increase in PTHrP mRNA in the unoccupied horn of a unilaterally pregnant rat could be elicited as rapidly as 1 h after balloon inflation and was maintained for up to 72 h. The same response was seen in uterine horns from virgin animals and could be reproduced by three different methods of imposing a physical stretch. Balloon-induced stretch also increased mRNA expression in a muscle bath system in vitro. Mechanotransduction appears to be largely, if not entirely, responsible for the preterm peak in PTHrP mRNA expression.

Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor.

In animal models, parathyroid hormone-related protein (PTHrP) increases placental calcium transport and inhibits contraction of uterine smooth muscle. The present studies were undertaken to characterize the expression of PTHrP in human uteroplacental tissues. PTHrP mRNA was identified by Northern analysis as a single species (approximately 1.8 kilobases) in human amnion, chorion, placenta, decidua, and myometrium. The most abundant signal was seen in amnion, where it was 10-400 times that in the other uteroplacental tissues. PTHrP mRNA abundance was decreased in amnion (but not in the other tissues) following the onset of labor (P less than 0.001). PTHrP mRNA in amnion appeared to be translated to a bioactive peptide, as PTHrP bioactivity and immunoreactive PTHrP in amnion correlated closely with PTHrP mRNA content (r = 0.86 and 0.95, respectively; P less than 0.05 and P less than 0.01). Amniotic fluid contained PTHrP, 21 +/- 6 pmol/liter (n = 10) at 16 weeks and 41 +/- 9 pmol/liter (n = 7) at 38 weeks (P = 0.05). These concentrations equaled or exceeded those found in plasma of patients with hypercalcemia secondary to PTHrP. After rupture of the fetal membranes, PTHrP mRNA in amnion was decreased by 78% (P less than 0.0001). This decrease appeared to be specific for PTHrP mRNA, as glyceraldehyde-3-phosphate dehydrogenase mRNA was unchanged following rupture of membranes. Like PTHrP mRNA, PTHrP bioactivity and immunoreactive PTHrP in amnion decreased significantly following rupture of membranes (P less than 0.03 and P less than 0.01, respectively). Since PTHrP is a potent antagonist of uterine muscle contraction, the decrease of PTHrP following rupture of the fetal membranes may play a key role in the onset of labor.

Endometrial venous aneurysms in three New Zealand white rabbits.

Hematuria in rabbits has been associated with uterine adenocarcinoma, uterine polyps, renal infarction, urolithiasis, cystitis, bladder polyps, and pyelonephritis. Three adult female New Zealand White rabbits (Oryctolagus cuniculus) developed apparent hematuria, as suggested by blood in their excreta pans. They had been immunized with antigen-adjuvant emulsions, but had uneventful clinical histories. Physical examination disclosed no abnormalities, and laboratory tests, including hematology, serum chemistries, urinalyses, urine cultures, ultrasonography, and intravenous pyelography disclosed mild anemia, hematuria, and proteinuria in two of the rabbits. Antibiotic therapy failed to alleviate clinical signs. Two rabbits were euthanized because of persistent urogenital bleeding and the third rabbit underwent exploratory laparotomy and ovariohysterectomy. Multiple endometrial venous aneurysms were present in the uteri of all rabbits and urogenital bleeding was attributed to episodic bleeding from these lesions. Varices and aneurysms of uterine subserosal and myometrial venous plexuses, but not of endometrial vessels in women have been reported. To our knowledge, endometrial venous aneurysms have not been reported in animals previously. Our findings indicate that the differential diagnoses for sporadic apparent hematuria in female rabbits should include endometrial aneurysms.