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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 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.

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.)

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25-dihydroxyvitamin D3, the generated osteoclasts expressed both PTHrP messenger RNA (mRNA) and protein. In addition, PTHrP was detected in the majority of actively resorbing osteoclasts in sections of newborn and adult mouse long bones. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in osteoclasts at active bone resorption sites as well as in actively synthesizing osteoblasts and bone lining cells. Localization of PTHrP was also demonstrated in osteoclast-like cells of human giant cell tumors from bone. In some of these tumors, a small proportion of the multinucleated cells expressed tartrate resistant acid phosphatase (TRAP), but not PTHrP mRNA or protein. Finally, both mRNA and protein for PTHrP were expressed in osteoclasts in sections of bone or joints from patients with Paget's disease, rheumatoid arthritis, and osteoarthritis. These observations raise the possibility that PTHrP might participate in osteoclast function.