Patrice E. Poubelle

Alendronate for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis

Background Osteoporosis is a common complication of long-term glucocorticoid therapy for which there is no well-proved preventive or restorative treatment. Methods We carried out two 48-week, randomized, placebo-controlled studies of two doses of alendronate in 477 men and women, 17 to 83 years of age, who were receiving glucocorticoid therapy. The primary end point was the difference in the mean percent change in lumbar-spine bone density from base line to week 48 between the groups. Secondary outcomes included changes in bone density of the hip, biochemical markers of bone turnover, and the incidence of new vertebral fractures. Results The mean (±SE) bone density of the lumbar spine increased by 2.1±0.3 percent and 2.9±0.3 percent, respectively, in the groups that received 5 and 10 mg of alendronate per day (P<0.001) and decreased by 0.4±0.3 percent in the placebo group. The femoral-neck bone density increased by 1.2±0.4 percent and 1.0±0.4 percent in the respective alendronate groups (P<0.01) and decre...

Two-year effects of alendronate on bone mineral density and vertebral fracture in patients receiving glucocorticoids: A randomized, double-blind, placebo-controlled extension trial

OBJECTIVE To evaluate the continued efficacy and safety of alendronate (ALN) for up to 2 years in patients receiving glucocorticoids. METHODS This is a 12-month extension of a previously completed 1-year trial of daily ALN, performed to evaluate the effects of ALN over a total of 2 years in 66 men and 142 women continuing to receive at least 7.5 mg of prednisone or equivalent daily. All patients received supplemental calcium and vitamin D. The primary end point was the mean percentage change in lumbar spine bone mineral density (BMD) from baseline to 24 months. Other outcomes included changes in hip and total body BMD, biochemical markers of bone turnover, radiographic joint damage of the hands, and vertebral fracture incidence. RESULTS The mean (+/-SEM) lumbar spine BMD increased by 2.8 +/- 0.6%, 3.9 +/- 0.7%, and 3.7 +/- 0.6%, respectively, in the groups that received 5 mg, 10 mg, and 2.5/10 mg of ALN daily (P < or = 0.001) and decreased by -0.8 +/- 0.6% in the placebo group (P not significant) over 24 months. In patients receiving any dose of ALN, BMD was increased at the trochanter (P < or = 0.05) and maintained at the femoral neck. Total body BMD was increased in patients receiving 5 or 10 mg ALN (P < or = 0.01). These 2 dose levels of ALN were more effective than placebo at all sites (P < or = 0.05). Bone turnover markers (N-telopeptides of type I collagen and bone-specific alkaline phosphatase) decreased 60% and 25%, respectively, during treatment with ALN (P < or = 0.05). There were fewer patients with new vertebral fractures in the ALN group versus the placebo group (0.7% versus 6.8%; P = 0.026). The safety profile was similar between treatment groups. CONCLUSION Alendronate is an effective, well-tolerated therapy for the prevention and treatment of glucocorticoid-induced osteoporosis, with sustained treatment advantages for up to 2 years.

Production of monoclonal antibodies against interleukin-1α and -1β: Development of two enzyme immunometric assays (EIA) using acetylcholinesterase and their application to biological media

We describe two series of monoclonal antibodies (mAbs) directed against human interleukin-1 alpha (36 mAbs) and -1 beta (11 mAbs). The binding compatibility of each of mAb was studied using biotin-labelled mAbs in immunometric tests. Among the different pairs of compatible mAbs, we selected one pair for each interleukin-1 (IL-1) with optimal properties for a two-site immunometric assay. In these assays, covalent conjugates of mAb coupled to the tetrameric form of acetylcholinesterase (mAb-AChE) were used as tracers. The tests were performed in 96-well microtiter plates coated with the complementary mAb. Both assays appeared sensitive and specific since minimum detectable concentrations as low as 1 pg/ml were determined for each IL-1 without any significant cross-reactivity (less than 0.01%). The intra-assay precision was also very good with a coefficient of variation of less than 10% over a wide range (between 3 and 500 pg/ml depending on the time devoted to the enzymatic reaction). The high sensitivity and precision of the assays can be ascribed to the high affinities of the mAbs as well as the optimal catalytic properties of AChE. The specificity of the determination performed in culture medium was demonstrated using different validation tests including a comparison with a bioassay and the fractionation of samples by molecular sieve chromatography. Evidence is presented that the assay could be used for the determination of IL-1 levels in biological media such as plasma or serum.

Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils

Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX‐2) in human neutrophils. A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM‐CSF, TNF‐α, and fMLP, induced COX‐2 protein expression through signaling pathways involving transcription and protein synthesis events. Northern blots showed that freshly isolated neutrophils expressed low levels of COX‐2 mRNA, which rapidly increased after incubation with inflammatory agents. A characterization of the signal transduction pathways leading to COX‐2 protein expression was initiated. In LPS‐treated neutrophils, efficient induction of COX‐2 required the presence of serum and involved ligand binding to the CD14 surface antigen. The specific inhibitor of p38 mitogen‐activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX‐2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types. Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX‐2, raising the possibility that phospholipase D activation might take part in the process of COX‐2 induction. Major COX‐2‐derived prostanoids synthesized by inflammatory neutrophils were identified by liquid‐chromatography and tandem mass‐spectrometry as TXA2 and PGE2. The agonist‐induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX‐2 inhibitor NS‐398. These results show that COX‐2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil. They support the concept that, in these cells, the COX‐2 isoform is preeminent over COX‐1 for the stimulated‐production of prostanoids, and also suggest that neutrophil COX‐2 displays a distinct profile of expression among circulatory cells.—Pouliot, M., Gilbert, C., Borgeat, P., Poubelle, P. E., Bourgoin, S., Créminon, C., Maclouf, J., McColl, S. R., Naccache, P. H. Expression and activity of prostaglandin endoperoxide synthase‐2 in agonist‐activated human neutrophils. FASEB J. 612, 1109–1123 (1998)

Surface RANKL of Toll-like receptor 4-stimulated human neutrophils activates osteoclastic bone resorption.

Inflammatory bone loss in septic and inflammatory conditions is due to increased activity of osteoclasts that requires receptor activator of NF-kappa B-ligand (RANKL). Neutrophils are the predominant infiltrating cells in these conditions. Although disease severity is linked to neutrophils, their role in evolution of bony lesions is not clear. We show that lipopolysaccharide (LPS), a toll-like receptor 4 ligand, up-regulated the expression of membrane RANKL in human blood neutrophils and murine air pouch-derived neutrophils. LPS-activated human and murine neutrophils, cocultured with human monocyte-derived osteoclasts and RAW 264.7 cells, respectively, stimulated bone resorption. Transfection of PLB-985 neutrophil-like cells with RANKL antisense RNA reduced osteoclastogenesis. Synovial fluid neutrophils of patients with exacerbation of rheumatoid arthritis strongly expressed RANKL and activated osteoclastogenesis in coculture systems. Osteoprotegerin, the RANKL decoy receptor, suppressed osteoclast activation by neutrophils from these different sources. Moreover, direct cell-cell contact between neutrophils and osteoclasts was visualized by confocal laser microscopy. Activation of neutrophil membrane-bound RANKL was linked to tyrosine phosphorylation of Src-homology domain-containing cytosolic phosphatase 1 with concomitant down-regulation of cytokine production. The demonstration of these novel functions of neutrophils highlights their potential role in osteoimmunology and in therapeutics of inflammatory bone disease.

Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: Implications for rheumatoid arthritis?

Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA1-3. Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA3 agonist l-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA1/3 receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-α (TNF-α) increases LPA3 mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-α-primed FLS is abolished by LPA1/3 receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA1/3 receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA1 and LPA3 may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.

The calcium-binding protein S100A12 induces neutrophil adhesion, migration, and release from bone marrow in mouse at concentrations similar to those found in human inflammatory arthritis.

We investigated the proinflammatory activities of S100A12 in the context of synovial inflammation. S100A12 levels were increased in the synovial fluids and plasma of patients with gout, rheumatoid arthritis, psoriatic arthritis, and undetectable in osteoarthritis, a noninflammatory disorder. S100A12 proved to induce neutrophil adhesion to fibrinogen via Mac-1 at concentrations similar to those found in the synovial fluids. Similar concentrations induced the recruitment of large numbers of neutrophils and monocytes in the murine air pouch model. To characterize the effect of increased S100A12 plasma levels, mice were injected intravenously with S100A12. This led to the mobilization of neutrophils from the bone marrow to the peripheral blood. These results suggest that S100A12 stimulates the accumulation of neutrophil by inducing their release from the bone marrow, as well as by activating their adhesion and migration toward inflammatory sites.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Scaffold-Guided Subchondral Bone Repair: Implication of Neutrophils and Alternatively Activated Arginase-1+ Macrophages

Background: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. Hypothesis: Chitosan–glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. Study Design: Controlled laboratory study. Methods: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (±blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37°C were analyzed by proteomics and neutrophil chemotaxis assays. Results: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1β or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (±blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. Conclusion and Clinical Relevance: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

Inflammatory Microcrystals Alter the Functional Phenotype of Human Osteoblast-Like Cells In Vitro: Synergism with IL-1 to Overexpress Cyclooxygenase-2

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE2 in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D3-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.