Partial hydroxylation of certain lysines in collagen.

Peptides derived from the al chain of collagen have been isolated in small amounts and have been shown to differ from ones found in expected amounts only by substitution of hydroxylysine for lysine. This observation indicates that hydroxylation of these lysines by protocollagen hydroxylase has been effected to a very minor extent.

Immunoglobulin M and secretory immunoglobulin A: presence of a common polypeptide chain different from light chains.

Unique polypeptide chains have been isolated from S-sulfonated light-chain fractions of human serum immunoglobulin M and colostral immunoglobulin A. Their electrophoretic mobilities, molecular weights, peptide maps, amino acid compositions, and antigenic determinants are very similar or perhaps identical but differ from those of light chains and secretory piece.

Effects of corticosteroids on immunity in man. I. Decreased serum IgG concentration caused by 3 or 5 days of high doses of methylprednisolone.

To study the effects of methylprednisolone on immune mechanisms in the absence of other immunosuppressive agents or immunologically mediated diseases, we gave 17 normal adult male volunteers 96 mg of methylprednisolone daily for 3-5 days and compared results with 12 untreated controls who were studied simultaneously, 86% of treated volunteers had significant decreases in the concentrations of serum IgG. 2-4 wk after methylprednisolone, the treated volunteers had a mean decrease in IgG of 22% compared with a decrease of only 1% in untreated controls. Likewise, significant decreases in IgA concentration occurred in 43% of treated volunteers, whereas significant decreases in IgM occurred in only 14%. The lowest immunoglobulin levels occurred during the 2nd wk after a 3 day course of methylprednisolone and during the 3rd wk after a 5 day course of drug. Slightly decreased plasma concentration of [(125)I]IgG was seen in six of seven volunteers who received a 5 day course but in only one of four who received a 3 day course of drug. However, an increase in the rate of plasma clearance of IgG occurred only during the treatment period itself. During the period when the serum concentration of IgG was falling, the specific activity of IgG in the serum was relatively higher in treated men than in controls indicating decreased entry of newly synthesized IgG into the circulation. These findings suggest that a short course of methylprednisolone treatment causes a pronounced and sustained decrease in serum IgG due to increased catabolism during drug administration and to decreased synthesis during and for a variable time after drug administration.

The mechanism of appearance of immunoglobulin A in nasal secretions in man.

Experiments were done to investigate mechanisms of appearance of both 7 S and 11 S IgA in nasal secretions. Three IgA preparations, (a) 11 S IgA from nasal secretions, (b) 7 S IgA from homologous serum, and (c) 7 S IgA from autologous serum, were isolated, labeled, and injected intravenously into volunteers. The rate of disappearance from plasma and the extent and nature of their appearance in nasal secretions were examined in detail. After intravenous injection, 11 S IgA from nasal secretions disappeared rapidly from plasma. However, only negligible amounts of 11 S IgA were detected in nasal secretion, which suggested that rapid selective secretion of circulating 11 S IgA does not occur as a mechanism of IgA accumulation in nasal secretions. Both the homologous and autologous 7 S IgA preparations disappeared from plasma at a normal rate, and both appeared in nasal secretions unchanged in sedimentation behavior. The specific activity of IgA in nasal secretions, when related to the total IgA concentration, was about 30-fold less than that in serum. When related to only the 7 S IgA concentration of nasal secretions, the specific activity was about one-half that of serum. These studies are consistent with the following hypotheses: (a) circulating 7 S IgA is a source of part of the 7 S IgA found in nasal secretions. The remainder of the nasal secretion 7 S IgA may be synthesized locally in the tissues of the upper respiratory tract; (b) 11 S IgA in nasal secretions is not assembled from serum 7 S IgA components; and (c) 11 S IgA in nasal secretions is synthesized de novo in the tissues of the upper respiratory tract.

Blastogenic suppression and alloantibody activity of sera from renal allograft recipients.

To evaluate whether immunological enhancement plays a role in adaptation to renal allografts, we studied sera from transplant recipients to determine whether those which suppressed mixed leukocyte culture (MLC) responses in vitro contained alloantibodies reactive with donor cells. Sera from five of nine renal transplant recipients consistently and specifically suppressed autologous autologous MLC responses to donor cells without impairing the blastogenic responses to third-party leukocytes, soluble antigens, or nonspecific mitogenic agents. In three of the five cases the suppressive activity of the serum was striking; in two cases the effect was less marked but still readily demonstrable in studies designed to evaluate the dose of serum which provided optimal suppression of MLC responses. Serum from one of the recipients nonspecifically suppressed blastogenic activity both to donor cells and other stimuli. No alloantibody reactive with donor leukocytes was found in any of the sera which exhibited suppressive activity in MLC, whereas in one patient, serum which contained antibody reactive with donor cells did not suppress MLC response to that donor. These findings suggest that, if the serum factors which suppress MLC responses in vitro are enhancing antibodies, they are not detectable even with very sensitive techniques either because they are present in very low concentrations, belong to immunoglobulin classes other than IgA, IgG, or IgM, or are complexed with donor antigen in such a way that their ability to react with fresh donor cells in vitro is blocked.

Non-collagenous proteins of rat dentin. Evidence that phosphoprotein is not covalently bound to collagen.

The non-collagenous proteins of rat dentin that remain firmly bound to the matrix after demineralization were studied in order to ascertain if they are covalently linked to insoluble dentin collagen. After solubilization with CNBr or with bacterial collagenase, unusually small amounts of dentin phosphoprotein were detected in the matrix. The phosphoprotein obtained by CNBr digestion of the matrix was separated from collagen peptides using two chromatographic steps. Thus even this small quantity of phosphoprotein found in decalcified rat dentin matrix was not covalently bound to collagen.

Evidence for several gamma-carboxyglutamic acid-containing proteins in dentin.

With anion-exchange chromatography, the gamma-carboxyglutamic acid (Gla)-containing proteins of rat dentin were separated into four closely related fractions (gamma 1-gamma 4). Edman degradation of gamma 2 gave two NH2-terminal sequences with a minor sequence beginning five residues shorter than the major one. Gel electrophoresis of gamma 2 yielded one major and one minor protein band. Fraction gamma 3 gave one band on gel electrophoresis and a single NH2-terminal sequence. The composition of gamma 4 suggested that, compared to gamma 2 and gamma 3, a portion of the COOH-terminal was missing. Thus some of the heterogeneity of rat dentin Gla-containing proteins can be explained by shortened ends.

Purification and some properties of the phosphoprotein from rat incisors.

The phosphoprotein of rat incisors has been purified by successive gel and ion-exchange chromatography. The product gave a single band on polyacrylamide gel electrophoresis and contained approximately 34% phosphoserine and 32% aspartic acid. Alkaline elimination experiments showed all the phosphate to be present as phosphoserine. Ultraviolet spectra in the presence or absence of ATP showed that the phosphoprotein did not contain an nucleotide moiety as suggested by Veis, A., Spector, A. R. and Zamoscianyk, H. ((1972) Biochim. Biophys. Acta 257, 404-413) for bovine dentin phosphoprotein.

Macrophages and mast cells control the neutrophil migration induced by dentin proteins.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1beta, TNF-alpha, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP-treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1beta, TNF-alpha, and MIP-2 by macrophages. This inhibitory activity of the DSP-stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.

Developmental analysis and computer modelling of bioengineered teeth.

Here we present the developmental progression of bioengineered pig teeth from 1 to 25 weeks of development. We demonstrate that 2-25 week implants contained embryonic tooth bud- and cap-stage tooth structures consisting of dental epithelium expressing the sonic hedgehog gene and condensed dental mesenchyme. Implants harvested at 18-25 weeks also contained tooth bud-like structures, as well as mature tooth structures containing enamel, dentin and pulp tissues. Immunohistochemical analyses confirmed the expression of dentin- and enamel-specific proteins in differentiated bioengineered tooth tissues. Three-dimensional computer modelling further demonstrated a spatial organization of enamel, dentin and pulp tissues resembling that of natural teeth. We conclude that bioengineered teeth commonly exhibit morphological stages characteristic of naturally forming teeth. Furthermore, the presence of immature tooth buds at all times assayed and increased numbers of bioengineered tooth structures over time suggests that porcine dental progenitor cells maintain the ability to form teeth for at least 25 weeks.

The nature and functional significance of dentin extracellular matrix proteins.

Odontoblasts are responsible for formation of predentin, which is transformed to dentin when apatite crystals are formed and the fibrillar matrix becomes mineralized. Odontoblasts are specialized cells that synthesize and secrete a unique set of non-collagenous proteins (NCPs), as well as the collagenous matrix largely comprised of type I collagen. The NCPs consist of dentin specific and mineralized tissue specific proteins, as well as other proteins that are found in a variety of tissues. Three dentin specific proteins have been recognized to date: dentin phosphoprotein (DPP), also called phosphophoryn, AG1 (dentin matrix protein 1, Dmp1) and dentin sialoprotein (DSP). DPP appears to be made by odontoblasts and appears at the mineralization front within a short time. It may be secreted via odontoblastic processes. DPP binds to collagen and potentially initiates formation of apatite crystals. A second DPP function appears to be to bind to the 100 face of growing apatite crystals and to inhibit or slow their growth; thus, DPP may play a dual role by initiating mineralization and then affecting the crystal growth and perhaps the habit of the crystals. Although no function has been ascribed to AG1 or DSP, they should prove to be important markers for the odontoblast phenotype. A recent unique finding is that two separate genes appear to code for more than one DSP mRNA; other transcripts may result from differential splicing. Examples of mineralized tissue specific proteins expressed by osteoblasts as well as odontoblasts are bone sialoprotein (BSP) and osteocalcin. Some NCPs expressed by osteoblasts, odontoblasts and several other tissues include osteopontin (OPN) and the chondroitin sulfate containing proteoglycans, decorin and biglycan. We propose that characterization of odontoblasts in tissues and cultures should rely upon utilization of sets of markers for the above NCPs and their mRNAs. Similar approaches are commonly used in investigations on osteoblasts. Finally, dentin (like bone) contains other molecules such as growth factors, and serum derived proteins, found within the matrix; no functional significance has yet been placed upon this finding. Future experiments should focus upon the elucidation of the three dimensional structures of the collagenous fibrillar network and of the NCPs to determine the relationships to mineralization. The role played by odontoblasts in controlling extracellular events, such as by selective secretory routes, will require careful exploration.

Evidence for the formation of a complex between osteopontin and osteocalcin.

We hypothesize that the mechanisms governing bone formation and remodeling involve the assembly of some of the components of the extracellular matrix into supramolecular complexes. We have examined the associations of osteopontin (OPN) with other proteins isolated from demineralized rat long bones. Three ligand binding techniques were used to demonstrate the formation of complexes between osteopontin and osteocalcin (OCN). Using gel overlay assays, the binding between soluble 125I-OPN and OCN immobilized in acrylamide gels was visualized. Competition for 125I-OPN-OCN complexes was demonstrated when unlabeled OCN-enriched bone extract was included in gel overlay solutions. Also, gel overlay assays showed 125I-OCN binding to OPN. Saturable binding was shown in solid-phase filter binding assays, which yielded an equilibrium binding constant of moderately high affinity (approximately 10(-8) M). Specificity of OPN-OCN complex formation was confirmed by measuring binding in the presence of unlabeled OPN and OCN versus a bone-localized serum protein, alpha 2HS-glycoprotein. Finally, the formation of soluble complexes were demonstrated in a modified Hummel-Dreyer gel filtration assay. These results indicate that OPN and OCN form complexes in vitro. The possible functions of OPN-OCN complexes in osteoclast recruitment and attachment are discussed.

Mechanism of fibroblast attachment to bone extracellular matrix: role of a 44 kilodalton bone phosphoprotein.

While the exact mechanisms regulating bone homeostasis are unknown, it is generally accepted that factors with the capacity to regulate cell attachment and spreading play a role in osteogenesis. A 44 kDa bone phosphoprotein (44K BPP), isolated from rat bone and synthesized by osteoblasts, was evaluated for its role in attachment and spreading of fibroblasts. In uncoated plates, enhanced cell attachment and spreading were observed when fibroblasts were exposed to the 44K BPP. The attachment properties of the bone phosphoprotein are different from those of fibronectin, in that the 44K BPP did not promote cell attachment in type I collagen wells, as was seen with fibronectin. Also, 44K BPP continued to enhance cell attachment up to 24 h, whereas cell attachment declined in time with cells exposed to fibronectin. Cycloheximide did not alter 44K BPP promotion of cell attachment, indicating that de novo protein synthesis was not required. These studies suggest that the 44K BPP is important in the regulation of cell attachment and spreading at sites of mineralization.

Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat.

Using immunohistochemical methods we studied the tissue localization of the extracellular matrix proteins osteopontin (OPN), osteocalcin (OC), and dentin sialoprotein (DSP) during the formation of acellular and cellular cementum in newly born rats. In the layer of acellular cementum of developing incisor and molar teeth we found a very strong staining for OPN but not for DSP or OC. Many cells immediately adjacent to acellular cementum and PDL cells were also positive for OPN but not for DSP or for OC. In contrast, cellular cementum in molar teeth stained strongly for OPN and OC but not for DSP. Consistent with these observations, the cells engaged in the formation of cellular cementum (cementoblasts and cementocytes) reacted strongly for OPN and OC but not for DSP. In advanced stages of dentinogenesis, both crown and root odontoblasts and dentin stained for OPN, OC, and DSP. Cells and matrices of surrounding alveolar bone stained for OPN and OC but not for DSP. We conclude that cementoblasts and cementocytes of cellular cementum produce OPN and OC but not DSP and thus express an osteoblast-like, not an odontoblast-like, phenotype. The cells responsible for the production of acellular cementum are likely cells of the PDL in close contact with the dental root surface. These fibroblast-like cells express OPN but not OC or DSP and accordingly express only a partial osteoblastic phenotype.

Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone.

The high-resolution, postembedding protein A-gold immunocytochemical technique was used to visualize the distribution of two noncollagenous bone proteins, osteopontin (OPN) and osteocalcin (OC), and two plasma proteins, alpha 2HS-glycoprotein (alpha 2HS-GP) and albumin (ALB), in sections of Lowicryl K4M-embedded rat tibial and alveolar bone. In the primary spongiosa of the metaphysis, a seam of organic material (lamina limitans) that labeled intensely with OPN and OC antibodies was observed at the bone/calcified cartilage interface just below the zone of vascular invasion of the growth plate. With deposition of bone matrix proper by osteoblasts in this region and its subsequent mineralization, extensive areas of bone were heavily labeled with anti-OPN, anti-OC, and anti-alpha 2HS-GP antibodies, where the majority of gold particles were associated with amorphous, electron-dense patches of organic material throughout the mineralized bone. In the unmineralized osteoid, substantially less labeling was observed, and where occasional mineralization loci were dispersed throughout the osteoid layer, these sometimes showed a concentration of gold particles. ALB labeling, on the other hand, was moderate and generally diffuse throughout the mineralized bone matrix and the osteoid. In alveolar bone, labeling patterns were generally similar to those found in tibial bone. Particularly striking in alveolar bone, however, was an intense anti-OPN labeling of (1) the lamina limitans at cell-lined bone surfaces, including that surrounding cell processes and osteocytes, (2) cement (reversal, resting) lines, and (3) the perilacumar matrix of some osteocytes. In summary, these data suggest that certain plasma proteins, such as alpha 2HS-GP, interact with bone matrix proteins, such as OPN and OC, at sites of tissue mineralization and that the presence of OPN in mineralized bone and at bone surfaces (lamina limitans) and cement lines has a multifunctional role, including regulation of mineralization and mediation of cell dynamics during endochondral and intramembranous bone modeling and remodeling.

A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone gamma-carboxyglutamic acid (Gla)-containing protein in osteoblasts.

Bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformaldehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.

Evidence that a non-RGD domain in rat osteopontin is involved in cell attachment.

The bone sialoprotein osteopontin (OPN) promotes cell attachment and spreading through its RGD (Arg-Gly-Asp) sequence. To study additional regions of OPN involved in cell attachment, peptides of rat OPN were evaluated for their capacity to mediate cell binding to wells in vitro. Human gingival fibroblasts were incubated on microtiter plates coated with either OPN or OPN peptides. A peptide of M(r) 28 kD, obtained after digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment to a similar degree as OPN. Sequence analysis showed that the amino terminus of the 28 kD peptide starts at Ser142 and therefore does not contain the RGD cell attachment sequence (residues 128-130). Cell attachment mediated through both OPN and the 28 kD peptide was blocked by the addition of GRGDSPA peptides or LM-609, a monoclonal antibody to the integrin alpha V beta 3, a receptor for vitronectin. A variant peptide, GRG-ESPA, did not alter cell attachment. Based on these observations, we conclude that (1) binding of OPN and the 28 kD peptide to fibroblasts involves binding to alpha V beta 3, (2) a site other than the RGD sequence on OPN is also involved in binding to integrins, and (3) the binding of this second site to alpha V beta 3 is inhibited by RGD-containing peptides.

Osteopontin posttranslational modifications, possibly phosphorylation, are required for in vitro bone resorption but not osteoclast adhesion.

Osteopontin (OPN), a phosphorylated bone matrix glycoprotein, is an Arg-Gly-Asp (RGD)-containing protein that interacts with integrins and promotes in vitro attachment of a number of cell types, including osteoclasts. Gene knockout experiments support the idea that OPN is important in osteoclastic activity. We hypothesize that posttranslational modifications (PTMs) of OPN can influence its physiological function. Previous studies have suggested that phosphorylation of OPN and bone sialoprotein (BSP) is necessary for promoting osteoclast adhesion. However, no reports have explored the importance of phosphoserines and other PTMs in OPN-promoted bone resorption. To study this question, we determined the activities of different forms of OPN and BSP in three in vitro assays: attachment of osteoclasts; formation of actin rings; and bone resorption. For each assay, cells were incubated for 4-24 h, in the presence or absence of RGDS or RGES peptides, to test the involvement of integrin binding. In addition to OPN, activities of milk OPN (fully phosphorylated) and recombinant OPN (rOPN, no phosphate) were compared. We purified two forms of OPN (OPN-2 and OPN-5), which differ in the level of phosphorylation, and compared their activities. For comparison, the activities of BSP and recombinant BSP (rBSP) were determined. All forms of OPN, including rOPN, significantly increased attachment of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. BSP and rBSP also promoted cell attachment. After 4 h of incubation, the proportion of cells with actin rings was increased with OPN, milk OPN, and BSP. In the presence of RGDS peptide, osteoclast retraction and the disruption of actin rings were observed, whereas no effect was seen with RGES. In the resorption assay, the number of pits and the total resorbed area per slice were increased in the presence of OPN, milk OPN, and BSP. As in other assays, the OPN enhancement of resorption was inhibited by RGDS, but not RGES, peptides. Significantly, rOPN and rBSP did not promote bone resorption. OPN-5 promoted resorption to a greater extent than OPN-2, and milk OPN significantly stimulated resorption to a greater extent than OPN. Our data suggest that: (1) the RGD sequence of OPN is essential in OPN-mediated cell attachment, actin ring formation, and bone resorption; and (2) some form of PTM, possibly phosphorylation, is necessary for in vitro osteoclastic bone resorption, but not for cell attachment and actin ring formation.

Specific in vitro binding of radiolabeled antibodies to cell surface antigens: interaction of antihuman thymocyte globulin with human target cells.

We have developed a highly sensitive and reproducible in vitro method to measure the rate and amount of binding of radiolabeled antibodies to target cells. We reduced non-specific binding of the antibodies to the target cells approx. 92% by removing protein aggregates from the antibody solutions. Using a specially designed cup that simultaneously allows washing and collection of target cells, cell loss was eliminated. This method was used to study the in vitro binding properties of the IgG fraction of anti-human thymocyte globulin (ATG) to nucleated human cells and to cells of other species. We found the initial rapid uptake of labeled ATG-IgG slowed with prolonged incubation. Incubation temperature and ATG concentration increased the rate of uptake. Sequential absorption studies indicated that initial uptake was due to rapidly binding antibodies. After these antibodies were removed, the rate of binding for antibodies that remained was several fold less than that of the antibodies removed by the initial absorption. Since temperature and the concentration of antibody and target cells can be rigidly controlled, this in vitro model system is ideally suited to quantify optimal conditions and kinetics of antibody binding to cell membrane antigens. Furthermore, the binding properties of antibody subpopulations in an antiserum may be determined by this technique. The maximum antibody binding capacity of various cell types can also be measured using the technique with a precision of +/- 14% on replicate determinations.

Immunolocalization of Gla proteins (osteocalcin) in rat tooth germs: comparison between indirect immunofluorescence, peroxidase-antiperoxidase, avidin-biotin-peroxidase complex, and avidin-biotin-gold complex with silver enhancement.

Odontoblasts and osteoblasts synthesize gamma-carboxyglutamatic acid (Gla)-containing proteins which are partially deposited in the mineralizing tissues and partially released into the plasma. Using four immunostaining techniques, we have evaluated the question of whether dentin Gla proteins (DGP) are transported to the mineralization front through the odontoblast processes. Undecalcified sections of rat incisors and molar tooth germs were immunostained with affinity-purified antibodies to DGP using the following methods: indirect immunofluorescence; peroxidase-antiperoxidase (PAP); avidin-biotin-peroxidase complex (ABC-peroxidase); and avidin-biotin-gold complex with silver enhancement (ABC-GSS). The results obtained with these four procedures were compared with respect to the developmental appearance of DGP, staining intensity and presence in odontoblastic processes, predentin, dentin, and blood vessels. Qualitatively, similar results were obtained with the four, with respect to the distribution and developmental appearance of DGP, with two exceptions: indirect immunofluorescence never stained DGP within blood vessels, whereas the other methods occasionally did; and because of its sensitivity, only the ABC-GSS method revealed immunostaining for DGP in odontoblastic processes. All methods revealed weak immunostaining in predentin which was considerably enhanced with hyaluronidase treatment; however, hyaluronidase only moderately increased predentin immunostaining with ABC-GSS. Of these four procedures, ABC-GSS is the most sensitive; however, ABC-GSS appears to detect predominantly antigens at the surface of tissue sections. We conclude that DGP is present in odontoblastic processes but in low amounts; the weak staining was due either to rapid transport of DGP through the process or to the fact that this mode of transport is limited.