Bone morphogenetic protein signaling regulates skin inflammation via modulating dendritic cell function.

BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-ß family that signal via the BMP receptor (BMPR) signaling cascade, distinct from canonical TGF-ß signaling. BMP downstream signaling is strongly induced within epidermal keratinocytes in cutaneous psoriatic lesions, and BMP7 instructs monocytic cells to acquire characteristics of psoriasis-associated Langerhans dendritic cells (DCs). Regulatory T (Treg)-cell numbers strongly increase during psoriatic skin inflammation and were recently shown to limit psoriatic skin inflammation. However, the factors mediating Treg-cell accumulation in psoriatic skin currently remain unknown. OBJECTIVE: We sought to investigate the role of BMP signaling in Treg-cell accumulation in psoriasis. METHODS: The following methods were used: immunohistology of patients and healthy controls; ex vivo models of Treg-cell generation in the presence or absence of Langerhans cells; analysis of BMP versus canonical TGF-ß signaling in DCs and Treg cells; and modeling of psoriatic skin inflammation in mice lacking the BMPR type 1a in CD11c+ cells. RESULTS: We here demonstrated a positive correlation between Treg-cell numbers and epidermal BMP7 expression in cutaneous psoriatic lesions and show that unlike Treg cells from healthy skin, a portion of inflammation-associated Treg cells exhibit constitutive-active BMP signaling. We further found that BMPR signaling licenses inflammation-associated Langerhans cell/DC to gain an enhanced capacity to promote Treg cells via BMPR-mediated CD25 induction and that this effect is associated with reduced skin inflammation. CONCLUSIONS: Psoriatic lesions are marked by constitutive high BMP7/BMPR signaling in keratinocytes, which instructs inflammatory DCs to gain enhanced Treg-cell-stimulatory activity. Locally secreted BMP7 can directly promote Treg-cell generation through the BMP signaling cascade.

Sequential BMP7/TGF-ß1 signaling and microbiota instruct mucosal Langerhans cell differentiation.

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However, the mechanisms involved in their in situ development remain unclear. Here, we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria, signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium, TGF-ß1 finalizes LC differentiation, and ALK5 is crucial to this process. Moreover, the local microbiota has a major impact on the development of mucosal LCs, whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-ß1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Production of a polyclonal antibody against osteogenic protein-1, and its role in the diagnosis of osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a progressive degenerative disorder of the articular cartilage. Available diagnostic radiography has been poorly associated with the progress and severity of this clinical disease. As osteogenic protein-1 (OP-1) has been identified as a bone morphogenetic protein with a major role in cartilage repair, we aimed to evaluate its potential role in the diagnosis of OA. METHODS: This was an experimental study conducted at the Department of Biochemistry, Sikkim Manipal Institute of Medical Sciences, India. Polyclonal antibodies (i.e. anti-OP-1[f]) were raised against OP-1 in mice, and subsequently used in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect the presence of OP-1 in the synovial fluids of 75 osteoarthritic patients. For the purpose of correlation, the radiographic assessments of the knees of the 75 patients were graded using the Kellgren-Lawrence scoring system. RESULT: The polyclonal antibody (i.e. anti-OP-1[f]) raised against OP-1 was able to detect the presence of OP-1 in the synovial fluids of all the osteoarthritic patients via sandwich ELISA. The level of the OP-1 was found to be much higher than the reference range and correlated positively with the severity of OA (r = 0.24; p = 0.04). CONCLUSION: Our study shows that the polyclonal antibody, anti OP-1(f), could be used for the immunodiagnosis of osteoarthritis via sandwich ELISA.

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration.

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.

Sequential injection immunoassay for human bone morphogenic protein-7 using an immunoreactor immobilized with anti-human bone morphogenic protein-7 antibody--CdSe/ZnS quantum dot conjugates.

The detection of human bone morphogenic protein-7 (BMP-7) was achieved using a sequential injection immunoassay (SIIA) system. The SIIA system is based on the binding between BMP-7 and anti-human BMP-7 (AbBMP7)-CdSe/ZnS quantum dot (QD) conjugates immobilized onto a glass disk or an optical fiber, using fluorescence detection at excitation and emission wavelengths of 470 nm and 580 nm, respectively. The AbBMP7-QD conjugates were prepared by conjugating anti-human BMP-7 antibody (AbBMP7) to hydrophilic CdSe/ZnS core/shell quantum dots (QDs). The SIIA system was fully automated using software written in the LabVIEW™ development environment. The analytical performance of the SIIA system was characterized with a number of variables such as carrier flow rate and elution buffer. Under partially optimized operating conditions, the SIIA system had a linear calibration graph at up to 10.0 ng mL(-1) BMP-7 (R(2)≥0.975) and a sample frequency of two samples per hour. The SIIA system with an optical fiber immunosensor was used to detect and quantify BMP-7 in spiked real samples obtained from a biological process with recoveries in the range of 95-102%.

Immunohistological localization of BMP-2, BMP-7, and their receptors in knee joints with focal cartilage lesions.

INTRODUCTION: Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. METHODS: Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. RESULTS: BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. CONCLUSIONS: BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

Bone morphogenetic proteins inhibit CD40L/IL-21-induced Ig production in human B cells: differential effects of BMP-6 and BMP-7.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily. TGF-ß can affect class switch recombination in human B cells, but whether BMPs also play a role have not been tested. We investigated the functional effects of exogenously added BMPs on CD27(-) naive and CD27(+) memory B cells from healthy donors. BMP-2, -4, -6 and -7 inhibited CD40L/IL-21-induced production of IgM, IgG and IgA. BMP-6 reduced Ig production by 70% in memory B cells and more than 55% in naive B cells, whereas the other BMPs were slightly less potent. We observed a striking difference in functional effects between the structurally similar BMP-6 and BMP-7, as BMP-6 mainly inhibited plasmablast differentiation, and BMP-7 mainly induced apoptosis. In memory B cells, BMP-6 upregulated expression of DNA-binding protein inhibitor genes, but potently inhibited CD40L/IL-21-induced upregulation of the transcription factor XBP1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells.

Natural antibodies against bone morphogenic proteins and interferons in healthy donors and in patients with infections linked to type-1 cytokine responses.

In patients receiving recombinant therapeutic proteins, the production of antibodies against the therapeutics is a rising problem. The antibodies can neutralize and interfere with the efficacy and safety of drugs and even cause severe side effects if they cross-react against the natural, endogenous protein. Various factors have been identified to influence the immunogenic potential of recombinant human therapeutics, including several patients' characteristics. In recent years, so-called naturally occurring antibodies against cytokines and growth factors have been detected in naive patients before start of treatment with recombinant human therapeutics. The role of naturally occurring antibodies is not well understood and their influence on production of anti-drug antibodies is not known. One might speculate that the presence of naturally occurring antibodies increases the likelihood of eliciting anti-drug antibodies once treatment with the corresponding recombinant therapeutic protein is started. We screened serum samples from 410 healthy controls and patients for auto-antibodies against bone morphogenetic proteins (BMPs) 2 and 7 and interferon (IFN)-α, -ß, and -γ in a new 3-step approach: rough initial screening, followed by competition and protein A/G depletion. Naturally occurring antibodies against these proteins were detected in 2% to 4% of the tested sera. Individuals who are 65 years or older had a slightly higher occurrence of naturally occurring antibodies. Auto-antibodies against BMP-7 and IFN-α were mainly comprised of IgM isotypes, and natural antibodies against BMP-2, IFN-ß, and -γ were mainly IgG. To ensure assay specificity, assays were also used to detect antibodies against BMP-7 in patients being treated with rhBMP-7 before and after surgical procedure. Fifty percent of the treated patients had persistent anti-BMP-7 antibodies over time. The 3-step approach provides an attractive tool to identify naturally occurring antibodies in naive patients.

Immunogenicity of osteogenic protein 1: results from a prospective, randomized, controlled, multicenter pivotal study of uninstrumented lumbar posterolateral fusion.

OBJECT: The aim in this study was to detect and quantify antibody responses against recombinant human osteogenic protein 1 (OP-1) and to compare these responses to patient clinical outcomes and safety information. METHODS: A controlled, open-label, randomized, prospective, multicenter pivotal study was performed in which patients with single-level Grade I or II degenerative lumbar spondylolisthesis (Meyerding classification) and spinal stenosis underwent decompression and uninstrumented posterolateral spinal arthrodesis. Three hundred thirty-six patients were randomized in a 2:1 fashion to receive either OP-1 Putty or autogenous iliac crest bone graft. Patients were evaluated at regular postoperative intervals for radiographic results, clinical outcomes, and safety parameters for more than 36 months. Serum samples were collected over this period and evaluated for the presence of anti­OP-1 antibodies and neutralizing activity by using a battery of in vitro binding assays (including enzyme-linked immunosorbent assay [ELISA]) and cell-based bioassays, respectively. RESULTS: Antibodies were predominantly seen in the OP-1­treated patients, although some responses were recorded preoperatively and in patients receiving autograft alone. Antibody production peaked in the 6-week to 3-month postoperative time frame and diminished thereafter. Neutralizing antibodies (Nabs) were detected at 1 time point at least in 25.6% of the patients treated with OP-1 Putty, but were not found in any patient following the 24-month postoperative time period. A single autograft patient (1.2%) also presented with OP-1 Nabs. An anti­OP-1 antibody status did not correlate with any measure of patient outcomes or adverse events. CONCLUSIONS: Recombinant human OP-1 (bone morphogenetic protein 7), like many recombinant human proteins, induces an immune response following its use as a bone graft alternative. This response was transient and diminished over time, and there was no statistical evidence to suggest an association between Nab status and any of the efficacy or safety criteria that were examined.

Immunogenicity of bone morphogenetic proteins.

OBJECT: The object of this paper is to review the immunogenicity of bone morphogenetic proteins (BMPs) and to compare the results of the immunogenicity characterization and clinical consequences between recombinant human (rh)BMP-2 and recombinant human osteogenic protein-1 (rhOP-1/BMP-7). METHODS: The immunogenicity of therapeutic proteins and its clinical effects were reviewed. The characteristics of BMPs were also described in terms of immunogenicity. The methods and results of antibody detection in various clinical trials of rhBMP-2 and rhOP-1 were compared, including the most recent studies using a systematic characterization strategy with both a binding assay and bioassay. RESULTS: Similar to all recombinant human proteins, rhBMPs induce immune responses in a select subgroup of patients. Adverse effects from this response in these patients, however, have not been reported with antibody formation to either rhBMP-2 or rhOP-1. Overall, the incidence of antibody formation was slightly higher in rhOP-1 trials than in rhBMP-2 trials. CONCLUSIONS: Although they occur in a subgroup of patients, the immune responses against rhBMPs have no correlation with any clinical outcome or safety parameter. Clinicians, however, must be aware of the potential complications caused by the immunogenicity of BMPs until more studies clearly elucidate their safety.