Diane Duryea

Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells.

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFβ-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFβ- and TGFβ+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFβ3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFβ3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRα+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFα or anti-IL-1 therapies

IntroductionRat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFα, IL-1β, and receptor activator of NF-κB ligand (RANKL). Anti-IL-1 or anti-TNFα therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.MethodsLewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFα inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (α1AGP), multiple cytokines) were measured in serum (day 14 post onset).ResultsArthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFα reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFα and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFα and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum α1AGP, IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFα and IL-17. Anti-TNFα reduced systemic α1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic α1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.ConclusionsAnti-TNFα or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.

A novel chordin-like BMP inhibitor, CHL2, expressed preferentially in chondrocytes of developing cartilage and osteoarthritic joint cartilage

We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently, CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.

The evolving systemic and local biomarker milieu at different stages of disease progression in rat adjuvant-induced arthritis.

IntroductionRats with adjuvant-induced arthritis (AIA) were necropsied on 14 occasions during preclinical, acute clinical and chronic clinical stages of AIA progression to characterize local (joint protein extracts) and systemic (serum) levels of mediators regulating inflammation and bone erosion in conjunction with lymphoid tissue-specific leukocyte kinetics.ResultsSystemic increases in alpha1 acid glycoprotein, tumor necrosis factor-α (TNFα), interleukin (IL)-17, transforming growth factor beta (TGFβ), and chemokine (C–C motif) ligand 2 (CCL2) together with local IL-1α/β and TGFβ enrichment and local lymphoid hyperplasia preceded the onset of clinical disease and joint damage. Systemic upregulation of TNFα, IL-6, IL-17, TGFβ, IL-18, CCL2, receptor activator of nuclear factor-κβ ligand (RANKL), and prostaglandin E2 during acute and/or chronic AIA coincided with systemic leukocytosis and CD4+ T cell increase in blood and spleen. In contrast, progression of joint erosions during clinical AIA was associated with intra-articular increases in IL-1α/β, IL-6, RANKL, IL-17, TGFβ, CCL2, and KC/GRO and also a dramatic decline in osteoprotegerin.ConclusionThese data indicate that systemic and local events in inflammatory arthritis are discrete processes, driven by multiple cellular and humoral mediators with distinct kinetic profiles.

The candidate neuroprotective agent artemin induces autonomic neural dysplasia without preventing peripheral nerve dysfunction.

Artemin (ART) signals through the GFRα—3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfrα—3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.

Novel erythropoiesis stimulating protein (darbepoetin alfa) alleviates anemia associated with chronic inflammatory disease in a rodent model

OBJECTIVE We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy. METHODS Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly. RESULTS Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing. CONCLUSION ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.

The evolving systemic and local biomarker milieu at different stages of disease progression in rat collagen-induced arthritis

Rats with collagen-induced arthritis (CIA) were necropsied on 14 occasions from 4 days after induction to 27 days after disease onset to evaluate the kinetics of local (joint protein extracts) and systemic (serum) levels of inflammatory and pro-erosive factors. Systemic increases in α1 acid glycoprotein and KC/GRO together with systemic and local enrichment of interleukin (IL)-1β, IL-6, CCL2, transforming growth factor (TGF)-β and elevated IL-1α and IL-18 in joint extracts preceded the onset of clinical disease. Systemic upregulation of IL-1β, IL-6, TGF-β CCL2, RANKL and prostaglandin E2 (PGE2) during acute and/or chronic CIA coincided with systemic leukocytosis and a CD4+ T-cell increase in blood and spleen. In contrast, progression of joint erosions during clinical CIA was associated with intra-articular increases in IL-1α/β, IL-6, IL-18, CCL2, KC/GRO and RANKL, and a dramatic decline in osteoprotegerin (OPG). These data indicate that systemic and local events in inflammatory arthritis can be discrete processes, driven by multiple cellular and humoral mediators with distinct temporospatial profiles.

Hematologic effects of stem cell factor (SCF) and leukemia inhibitory factor (LIF) in vivo: LIF-induced thrombocytosis in SCF-primed mice

Abstract: Stem cell factor (SCF) administered as daily bolus injections in dose‐response experiments in mice causes a progressive and dramatic dose‐dependent panleukocytosis characterized by neutrophilia, eosinophilia, monocytosis, and lymphocytosis. SCF causes circulating platelet numbers to be dose‐dependently increased after 2 weeks of daily injections. Leukemia inhibitory factor (LIF) administered as daily bolus injections in mice causes a peripheral leukopenia that is largely due to peripheral lymphopenia. LIF causes thrombocytosis peaking after approximately 1 w. Coinjection of SCF and LIF for 1 to 2 wk in mice does not cause a much greater thrombocytosis than the maximum thrombocytosis achievable with SCF or LIF alone. On the other hand, daily injection of SCF for 5 days followed by daily injection of LIF for 5 to 6 d in mice causes a very substantial increase in platelets that was lineage‐specific in terms of not being accompanied by a generalized leukocytosis. In contrast, only a very modest thrombocytosis was noted in SCF‐primed LIF‐treated rats. LIF causes a large increase in the cytoplasmic volume of splenic megakaryocytes in mice, but not in rats. In conclusion, SCF‐induced priming followed by LIF‐induced maturation of megakaryocytes causes a substantial selective increase in the numbers of circulating platelets in mice.

IL‐1β and TNF‐α Produce Divergent Acute Inflammatory and Skeletal Lesions in the Knees of Lewis Rats

Arthritis is initiated and sustained by the release of myriad pro-inflammatory cytokines.1 In patients with rheumatoid arthritis, the dominant pro-inflammatory cytokines are interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α).2 Inhibition of IL-1β and/or TNF-α reduces the extent of inflammation in rheumatoid arthritis3,4 and in animal models of arthritis.5–7 Therefore, biologic agents that inhibit the action of these two cytokines are gaining rapid acceptance as early, aggressive treatments for rheumatoid arthritis. IL-1β and TNF-α act synergistically to induce and maintain inflammation and skeletal erosion in arthritis.1 The present study was designed to explore the early effects (48 h) by direct intra-articular injection of IL-1β or TNF-α. Our data show that both cytokines can initiate disease, but that IL-1β appears to play a more substantial role than TNF-α in the early stages of arthritis, particularly with respect to the induction of osseous lesions.

OP0016 Osteoclast numbers in joints of rats with adjuvant arthritis are decreased by treatment with il-1ra and/or peg stnf-ri

Background Adjuvant arthritis (AdA) induced in Lewis rats by injection of mycobacteria in oil is characterised by marked inflammation and skeletal damage. Recent work by our group using the AdA model has shown that skeletal destruction is mediated by the osteoclast differentiation factor osteoprotegerin (OPG) ligand,1 and that inhibition of bone loss is associated with anti-inflammatory efficacy.2 Objectives The current study was performed to assess whether anti-inflammatory molecules act to spare bone by regulating intra-lesional numbers of osteoclasts. Methods AdA was induced in male Lewis rats (n = 6/group) on Day 0, and hind paw swelling developed on Day 9. Animals were treated for 7 days (Days 9–15) and then necropsied (Day 16). Therapy consisted of interleukin-1 receptor antagonist (IL-1ra) or pegylated soluble tumour necrosis factor receptor type I (PEG sTNF-RI), administered alone or in all possible combinations, at the following doses: IL-1ra, 0.2, 1.0 or 5.0 mg/kg/hr (by s.c. infusion); PEG sTNF-RI, 0.25, 1.0 or 4.0 mg/kg/day (s.c bolus). Osteoclasts were detected by an indirect immunohistochemical method for cathepsin K. Inflammation, bone erosion and osteoclast numbers were scored blindly using tiered, semi-quantitative grading scales. Results Dose-dependent reductions in inflammation, bone erosion and osteoclast scores were observed for high doses of both anti-cytokine therapies, whether given alone or in combination. Conclusion These data indicate that anti-inflammatory doses of IL-1ra and PEG sTNF-RI protect the structural integrity of arthritic joints by regulating osteoclast numbers. References Kong, et al. Nature 1999;402:304 Feige, et al. Cell Mol Life Sci. 2000;57:1457