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NKR-P1B is a homodimeric type II transmembrane C-type lectinlike receptor that inhibits natural killer (NK) cell function upon interaction with its cognate C-type lectin-related ligand, Clr-b. The NKR-P1B:Clr-b interaction represents a major histocompatibility complex class I (MHC-I)-independent missing-self recognition system that monitors cellular Clr-b levels. We have generated NKR-P1B(B6)-deficient (Nkrp1b(-/-)) mice to study the role of NKR-P1B in NK cell development and function in vivo. NK cell inhibition by Clr-b is abolished in Nkrp1b(-/-) mice, confirming the inhibitory nature of NKR-P1B(B6). Inhibitory receptors also promote NK cell tolerance and responsiveness to stimulation; hence, NK cells expressing NKR-P1B(B6) and Ly49C/I display augmented responsiveness to activating signals vs NK cells expressing either or none of the receptors. In addition, Nkrp1b(-/-) mice are defective in rejecting cells lacking Clr-b, supporting a role for NKR-P1B(B6) in MHC-I-independent missing-self recognition of Clr-b in vivo. In contrast, MHC-I-dependent missing-self recognition is preserved in Nkrp1b(-/-) mice. Interestingly, spontaneous myc-induced B lymphoma cells may selectively use NKR-P1B:Clr-b interactions to escape immune surveillance by wild-type, but not Nkrp1b(-/-), NK cells. These data provide direct genetic evidence of a role for NKR-P1B in NK cell tolerance and MHC-I-independent missing-self recognition., (© 2015 by The American Society of Hematology.)

MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Thalassemia is an inherited disorder of alpha or beta globin chain synthesis leading to ineffective erythropoiesis requiring chronic transfusion therapy in its most severe form. This leads to iron overload, marrow expansion, and hormonal complications, which are implicated in bone deformity and loss of bone mineral density (BMD). In this 19-year retrospective longitudinal study, the relationships between BMD (determined by dual-energy X-ray absorptiometry) and risk factors for osteoporosis in 277 subjects with transfusion-dependent thalassemia were examined. The mean age at first review was 23.2 ± 11.9 years and 43.7% were male. Hypogonadism was present in 28.9%. Fractures were confirmed in 11.6% of subjects and were more frequent in males (16.5%) compared with females (7.7%). Lumbar spine (LS), femoral neck (FN), and total body (TB) Z-scores were derived. Patients with transfusion-dependent thalassemia had a significant longitudinal decline in BMD at the FN and TB. In the linear mixed-model analysis of BMD and risk factors for bone loss, FN Z-score was more significantly associated with risk factors compared with the LS and TB. The rate of decline at the FN was 0.02 Z-score per year and was 3.85-fold greater in males. The decline in FN Z-score over the last 5 years (years 15 to 19) was 2.5-fold that of the previous 7 years (years 8 to 14) and coincided with a change in iron chelator therapy from desferrioxamine to deferasirox. Hemoglobin (Hb) levels positively correlated with higher TB and LS Z-scores. In conclusion, the FN is the preferred site for follow-up of BMD. Male patients with β-thalassemia experienced a greater loss of BMD and had a higher prevalence of fractures compared with females. Transfusing patients (particularly males) to a higher Hb target may reduce the decline in BMD. Whether deferasirox is implicated in bone loss warrants further study., (© 2014 American Society for Bone and Mineral Research.)

Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.

Osteoclast inhibitory lectin (OCIL) is a type II C-type lectin and binds NK cell-associated receptor Nkrp1d and sulfated glycosaminoglycans. OCIL is expressed by several cell types found in bone and inhibits osteoclast differentiation. To determine whether OCIL may have wider effects on bone metabolism, we examined the effects of recombinant soluble OCIL on cultured osteoblasts and pre-osteoblastic KUSA O cells. Although OCIL did not affect osteoblast proliferation or apoptosis, or the formation of alkaline phosphatase positive colonies in cultured bone marrow, OCIL profoundly inhibited mineralization by primary osteoblasts and KUSA O cells in vitro. Analysis of ascorbate-treated KUSA O cells showed that addition of OCIL reduced bone sialoprotein (BSP), osterix and osteocalcin mRNA expression, as well as alkaline phosphatase activity while, in contrast, expression of markers associated with the earlier stages of osteoblast maturation or the transcription factors Runx2, ATF4 and c-fos were not affected by OCIL treatment. Indeed, osteocalcin expression was strongly inhibited within 3 days in a dose-dependent manner, although after subsequent removal of OCIL, osteocalcin mRNA levels recovered within 4 days. OCIL treatment also reduced osteocalcin expression in BMP-2 stimulated C2C12 cells. In support of a role for OCIL in mineralization, OCIL anti-sense oligonucleotide treatment of KUSA O cells increased mineralization and osteocalcin expression. In addition, insulin-, dexamethasone- and IBMX-stimulated KUSA O cells undergo adipocyte differentiation and OCIL treatment greatly suppressed this process. Consistent with this, OCIL also reduced adiponectin and resistin mRNA expression in these cells. Our data indicate that OCIL reduces osteoblastic function in vitro and this may be due to an inhibitory effect on osteoblast maturation. In addition, the reduction of adipocyte formation in KUSA O cells by OCIL indicates that OCIL may have wider effects on the mesenchymal lineage that may be important for both bone metabolism and other connective tissue functions.

Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide range of fetal and adult tissues, where it is thought to play a role in the regulation of angiogenesis during development. The temporal expression of PEDF during endochondral bone formation has not previously been reported. In this study, we analysed the expression pattern of PEDF in growing mouse hindlimbs from newborn day one through to maturation at week 9, using immunohistochemistry and in situ hybridization. PEDF expression was demonstrated in chondrocytes within the resting, proliferative and upper hypertrophic zones of the epiphyseal growth plate. The pattern of expression was consistent throughout the developmental stages of the mouse. In addition, PEDF was expressed by osteoblasts lining the bone spicules in the ossification zone of metaphyseal bone, as well as by osteoblasts lining cortical periosteum. These novel results demonstrate that PEDF is developmentally expressed in both cartilage and bone cells during endochondral bone formation, and strongly suggest that it may play a regulatory role in the processes of chondrocyte and osteoblast differentiation, endochondral ossification, and bone remodelling during growth and development of long bones.

Bone is a metabolically active and highly organized tissue consisting of a mineral phase of hydroxyapatite and amorphous calcium phosphate crystals deposited in an organic matrix. Bone has two main functions. It forms a rigid skeleton and has a central role in calcium and phosphate homeostasis. The major cell types of bone are osteoblasts, osteoclasts and chondrocytes. In the laboratory, primary cultures or cell lines established from each of these different cell types provide valuable information about the processes of skeletal development, bone formation and bone resorption, leading ultimately, to the formulation of new forms of treatment for common bone diseases such as osteoporosis.

Osteoclast inhibitory lectin (OCIL) is a membrane-bound C-type lectin that blocks osteoclast differentiation and, via binding to its cognate receptor NKRP1D, inhibits natural killer cell-mediated cytotoxicity. OCIL is a member of the natural killer cell receptor C-type lectin group that includes CD69 and NKRP1D. We investigated carbohydrate binding of soluble recombinant human and mouse OCIL in enzyme-linked immunosorbent assay-based assays. OCIL bound immobilized high molecular weight sulfated glycosaminoglycans, including fucoidan, lambda-carrageenan, and dextran sulfate, but not unsulfated dextran or sialated hyaluronic acid. Carbohydrate binding was Ca(2+)-independent. Binding of immobilized low molecular weight glycosaminoglycans, including chondroitin sulfate (A, B, and C forms) and heparin, was not observed. However, the soluble forms of these low molecular weight glycosaminoglycans competed for OCIL binding of immobilized fucoidan (as did soluble fucoidan, dextran sulfate, and lambda-carrageenan), indicating that OCIL does recognize these carbohydrates. Inhibition constants for chondroitin sulfate A and heparin binding were 380 and 5 nm, respectively. Immobilized and soluble monosaccharides did not bind OCIL. The presence of saturating levels of fucoidan, dextran sulfate, and lambda-carrageenan did not affect OCIL inhibition of osteoclast formation. The fucoidan-binding lectins Ulex europaeus agglutinin I and Anguilla anguilla agglutinin did not block osteoclast formation or affect the inhibitory action of OCIL. Although the osteoclast inhibitory action of OCIL is independent of sugar recognition, we have found that OCIL, a lectin widely distributed, but notably localized in bone, skin, and other connective tissues, binds a range of physiologically important glycosaminoglycans, and this property may modulate OCIL actions upon other cells.

Unlabelled: Osteoclast inhibitory lectin (OCIL) is a newly recognized inhibitor of osteoclast formation. We identified a human homolog of OCIL and its gene, determined its regulation in human osteoblast cell lines, and established that it can inhibit murine and human osteoclast formation and resorption. OCIL shows promise as a new antiresorptive., Introduction: Murine and rat osteoclast inhibitory lectins (mOCIL and rOCIL, respectively) are type II membrane C-type lectins expressed by osteoblasts and other extraskeletal tissues, with the extracellular domain of each, expressed as a recombinant protein, able to inhibit in vitro osteoclast formation., Materials and Methods: We isolated the human homolog of OCIL (hOCIL) from a human fetal cDNA library that predicts a 191 amino acid type II membrane protein, with the 112 amino acid C-type lectin region in the extracellular domain having 53% identity with the C-type lectin sequences of rOCIL and mOCIL. The extracellular domain of hOCIL was expressed as a soluble recombinant protein in E. coli, and its biological effects were determined., Results and Conclusions: The hOCIL gene is 25 kb in length, comprised of five exons, and is a member of a superfamily of natural killer (NK) cell receptors encoded by the NK gene complex located on chromosome 12. Human OCIL mRNA expression is upregulated by interleukin (IL)-1alpha and prostaglandin E2 (PGE2) in a time-dependent manner in human osteogenic sarcoma MG63 cells, but not by dexamethasone or 1,25 dihydroxyvitamin D3. Soluble recombinant hOCIL had biological effects comparable with recombinant mOCIL on human and murine osteoclastogenesis. In addition to its capacity to limit osteoclast formation, OCIL was also able to inhibit bone resorption by mature, giant-cell tumor-derived osteoclasts. Thus, a human homolog of OCIL exists that is highly conserved with mOCIL in its primary amino acid sequence (C-lectin domain), genomic structure, and activity to inhibit osteoclastogenesis.

We have identified two novel type II membrane-bound C-lectins, designated mOCILrP1 and mOCILrP2, of 218 and 217 amino acids, respectively, that share substantial identity with the murine osteoclast inhibitory lectin (OCIL). The extracellular domains of mOCILrP1 and mOCILrP2 share 83 and 75% identity, respectively, with the extracellular domain of mOCIL. When the extracellular domains were expressed as recombinant proteins, each inhibited osteoclast formation in murine bone marrow cultures treated with M-CSF and RANKL with similar potencies to mOCIL (IC(50) of 0.2 ng/ml). Distinct but highly related genes encoded the three OCIL family members, with mOCIL and mOCILrP2 controlled by an inverted TATA promoter, and mOCILrP1 by a TTAAAA promoter. However only mOCIL was robustly regulated by calciotropic agents, while mOCILrP1 was not expressed, and mOCILrP2 was constitutively expressed in osteoblasts. Immunohistochemistry using antipeptide antibodies to the intracellular domain of mOCILrP1/mOCILrP2 and to mOCIL demonstrated that mOCIL and mOCILrP1/mOCILrP2 were concordantly expressed in osteoblasts, chondrocytes, and in extraskeletal tissues. Further, their cellular distribution was identical to that of RANKL. The identification of three distinct genes that were functionally related implies redundancy for OCIL, and their concordant expression with that of RANKL suggests that the RANKL:OPG axis may be further influenced by OCIL family members.

We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin. In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect. The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor. Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells. mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase. Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid. In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues. The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.

Objective: To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA)., Methods: After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe., Results: TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion., Conclusion: Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.

Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1alpha,25(OH)(2)D(3), PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-kappaB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitro is unknown. Human PBMC-derived T cells, extracted using alphaCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fide osteoclasts were formed in vitro with activated T cells: IL-1alpha and TGFbeta further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3(+) T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis., (Copyright 1999 Academic Press.)

Parathyroid hormone-related protein is produced by many normal tissues including the skin, where it regulates growth and differentiation of keratinocytes. To define better the role of parathyroid hormone-related protein in the skin, we investigated the spatial and temporal expression of parathyroid hormone-related protein and mRNA by immunohistochemistry and in situ hybridization during the healing of skin wounds, and the effects of topical administration of a parathyroid hormone-related protein agonist [parathyroid hormone-related protein (1-36)] and a parathyroid hormone-related protein antagonist [parathyroid hormone (7-34)] on the healing rate and morphology of the wounds. Wounds were produced on the back of guinea pigs with a 4 mm punch, and wound sites were collected at different time points during the healing process. Parathyroid hormone-related protein was expressed in normal skin by all viable keratinocyte layers, hair follicles, and adnexae. Following injury, migratory keratinocytes at wound margins and the newly restored epidermis expressed increased levels of parathyroid hormone-related protein. The remodeling phase was associated with progressive restoration of the pattern of parathyroid hormone-related protein expression in normal epidermis. Granulation tissue myofibroblasts and infiltrating macrophages also expressed parathyroid hormone-related protein. In vitro studies using THP-1 cells (a promonocytic cell line) confirmed that macrophages expressed parathyroid hormone-related protein, especially after activation. Topical application of parathyroid hormone related protein (1-36) or parathyroid hormone (7-34) did not result in significant changes in the healing rate and morphology of the wounds. These findings demonstrated that, in addition to keratinocytes, myofibroblasts and macrophages also represent sources of parathyroid hormone-related protein during the healing of skin wounds. Although the data suggest a role for parathyroid hormone-related protein in the healing of skin and in the restoration of epidermal homeostasis, parathyroid hormone-related protein does not appear to be required for proper re-epithelialization in response to injury, potentially because of redundancy in epidermal growth and wound healing, as has been shown for other paracrine and autocrine growth factors of the epidermis.

The rat homeobox gene, rHox, was cloned from a rat osteosarcoma cDNA library. Southwestern and gel mobility shift analyses showed that rHox binds to the promoter regions of collagen (alpha1)I and osteocalcin genes while transient transfection with rHox resulted in repression of their respective promoter activities. In situ hybridization studies showed that rHox mRNA was widely expressed in osteoblasts, chondrocytes, skeletal muscle, skin epidermis, and bronchial and intestinal epithelial cells, as well as cardiac muscle in embryonic and newborn mice. However in 3-month-old mice, rHox mRNA expression was restricted to osteoblasts, megakaryocytes, and myocardium. Bone morphogenetic protein 2, a growth factor that commits mesenchymal progenitor cells to differentiate into osteoblasts, down-regulated rHox mRNA expression by 40-50% in UMR 201, a rat preosteoblast cell line, in a time- and dose-dependent manner. In contrast, PTH-related protein (PTHrP), recently shown to be a negative regulator of chondrocyte differentiation, significantly enhanced rHox mRNA expression in UMR 106-06 osteoblastic cells by 3-fold at 24 h while at the same time down-regulating expression of pro-alpha1(I) collagen mRNA by 60%. Expression of rHox mRNA in calvarial osteoblasts derived from PTHrP -/- mice was approximately 15% of that observed in similar cells obtained from normal mice. In conclusion, current evidence suggests that rHox acts as a negative regulator of osteoblast differentiation. Furthermore, down-regulation of rHox mRNA by bone morphogenetic protein 2 and its up-regulation by PTHrP support a role of the homeodomain protein, rHox, in osteoblast differentiation.

Multiple myeloma frequently leads to complications, such as osteolytic lesions, hypercalcemia, and pathological fractures. Increased bone resorption in myeloma is due to osteoclast activation. The nature of the osteoclast activator(s) remains unclear. We describe a case of multiple myeloma with marked hypercalcemia and skeletal complications that progressed rapidly despite chemotherapy. The patient had marked hypercalcemia at diagnosis (4.5 mmol/l), and elevated parathyroid hormone-related protein (PTHrP) levels were found in plasma. Analysis of the bone marrow trephine biopsy showed PTHrP gene transcription and protein in myeloma cells. These results provide strong evidence for the production of significant amounts of PTHrP by human myeloma cells. PTHrP has been measured as elevated in the plasma of patients with myeloma and might be an important contributor to the skeletal complications in this disease.

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the epidermis, where it is thought to play a role in the regulation of keratinocyte growth and differentiation. Most in vitro studies of normal keratinocytes use monolayer cell cultures, which have limitations, including the inability to reproduce the stratified structure of the epidermis. The objective of this study was to investigate PTHrP production and secretion, and mRNA expression in skin organotypic cultures. The cultures consisted of an artificial dermis with differentiating keratinocytes grown at the air-liquid interface. Immunohistochemical assessment of cytokeratins 14 and 10/13, involucrin, and proliferative cell nuclear antigen (PCNA) demonstrated that keratinocytes differentiated in a manner similar to keratinocytes in normal epidermis. PTHrP expression was demonstrated in all viable layers of the epidermis, as well as in some fibroblasts of the collagen lattice by immunohistochemistry and in situ hybridization. Since most fibroblasts expressed alpha-smooth muscle actin, these cells were interpreted to be consistent with myofibroblasts. PTHrP expression by myofibroblasts suggests a possible role for PTHrP in the regulation of contractibility of these cells. PTHrP was also detected in conditioned media for 50 days. In conclusion, because of its superior tissue morphology and ability to induce organized keratinocyte differentiation, this culture system will be an excellent model to study the role of PTHrP in pathologic and physiologic processes involving the epidermis in vitro.