Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Rα), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Rα ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Rα or Clf bolsters the hypothesis that Clc participates in Cntf-II.

INTRODUCTION:Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFalpha, IL-1beta, and receptor activator of NF-kappaB ligand (RANKL). Anti-IL-1 or anti-TNFalpha 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.METHODS:Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFalpha 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 (alpha1AGP), multiple cytokines) were measured in serum (day 14 post onset).RESULTS:Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFalpha 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-TNFalpha 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 antiTNFalpha 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 alpha1AGP, IL-1beta, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFalpha and IL-17. Anti-TNFalpha reduced systemic alpha1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic alpha1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.CONCLUSIONS:Anti-TNFalpha 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.

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 alpha1 acid glycoprotein and KC/GRO together with systemic and local enrichment of interleukin (IL)-1beta, IL-6, CCL2, transforming growth factor (TGF)-beta and elevated IL-1alpha and IL-18 in joint extracts preceded the onset of clinical disease. Systemic upregulation of IL-1beta, IL-6, TGF-beta CCL2, RANKL and prostaglandin E(2) (PGE(2)) 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-1alpha/beta, 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.

Background and aims: Transfer of CD4(+)CD45RB(Hi) T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process.Methods: CD4(+)CD45RB(Hi) or CD4(+)CD45RB(Lo) T cells (4x10(5)) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis.Results: Mice that received CD4(+)CD45RB(Hi) T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4(+)CD45RB(Lo) T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4(+)CD45RB(Hi) or CD4(+)CD45RB(Lo) T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4(+)CD45RB(Hi) T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4(+)CD45RB(Hi) T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor alpha which was unaffected by treatment with Fc-OPG.Conclusions: CD4(+)CD45RB(Hi) T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

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.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synergistically induce and sustain arthritis. Two competing hypotheses of arthritis induction are 1) that TNF preferentially mediates inflammation, whereas IL-1 impels bone destruction, or 2) that either cytokine controls the entire process. In this study, these propositions were tested in two experiments by instilling IL-1beta or TNF-alpha into one knee of Lewis rats (n = 6/group) to incite arthritis, after which semiquantitative scores for inflammation, bone resorption, osteoclasts, and cartilage integrity were acquired. In the induction study, IL-1beta or TNF-alpha (3, 10, or 30 micro g) was given once to incite arthritis. After 2 days, IL-1beta induced significant, dose-dependent increases in inflammation (mild to marked), bone resorption (minimal to moderate), and osteoclasts (minimal to moderate). In contrast, TNF-alpha induced minimal to mild inflammation but had little impact on resorption or osteoclasts. Both IL-1 and TNF (>/=10 micro g) yielded mild cartilage degeneration. Most lesion scores in TNF-treated rats were significantly lower than those in animals given the same dose of IL-1beta. In the persistence study, rats were injected once with IL-1 or TNF (10 micro g) and maintained for 2, 3, or 7 days. IL-1beta significantly enhanced inflammation (all 3 days), bone resorption (days 2 and 3), osteoclasts (days 2 and 3), and cartilage matrix loss (days 2 and 3), whereas TNF-alpha augmented inflammation (days 2 and 3) and cartilage degeneration (day 2) but not bone resorption or osteoclasts. Thus, both IL-1beta and TNF-alpha can launch inflammation, but IL-1beta drives skeletal destruction.

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.

Annals of the New York Academy of SciencesVolume 987, Issue 1 p. 295-298 IL-1β and TNF-α Produce Divergent Acute Inflammatory and Skeletal Lesions in the Knees of Lewis Rats GIUSEPPE CAMPAGNUOLO, GIUSEPPE CAMPAGNUOLO Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USA G.C. and B.B. are equal contributors.Search for more papers by this authorBRAD BOLON, BRAD BOLON Department of Pathology, Amgen Inc., Thousand Oaks, California 91320-1789, USA G.C. and B.B. are equal contributors.Search for more papers by this authorL.I. ZHU, L.I. ZHU Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorDIANE DURYEA, DIANE DURYEA Department of Pathology, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorULRICH FEIGE, ULRICH FEIGE Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorDEBRA ZACK, Corresponding Author DEBRA ZACK Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USAAddress for correspondence: Dr. Debra Zack, Amgen, One Amgen Center Drive, M/S 29-M-B, Thousand Oaks, CA 91320-1799. Voice: 805-447-6684; fax: 805-480-1329. dzack@amgen.comSearch for more papers by this author GIUSEPPE CAMPAGNUOLO, GIUSEPPE CAMPAGNUOLO Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USA G.C. and B.B. are equal contributors.Search for more papers by this authorBRAD BOLON, BRAD BOLON Department of Pathology, Amgen Inc., Thousand Oaks, California 91320-1789, USA G.C. and B.B. are equal contributors.Search for more papers by this authorL.I. ZHU, L.I. ZHU Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorDIANE DURYEA, DIANE DURYEA Department of Pathology, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorULRICH FEIGE, ULRICH FEIGE Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USASearch for more papers by this authorDEBRA ZACK, Corresponding Author DEBRA ZACK Department of Inflammation, Amgen Inc., Thousand Oaks, California 91320-1789, USAAddress for correspondence: Dr. Debra Zack, Amgen, One Amgen Center Drive, M/S 29-M-B, Thousand Oaks, CA 91320-1799. Voice: 805-447-6684; fax: 805-480-1329. dzack@amgen.comSearch for more papers by this author First published: 24 January 2006 https://doi.org/10.1111/j.1749-6632.2003.tb06066.x Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume987, Issue1IMMUNE MECHANISMS AND DISEASEApril 2003Pages 295-298 RelatedInformation

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRalpha displayed chondrogenic activity in the presence of TGFbeta3 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. TGFbeta-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 TGFbeta- and TGFbeta+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFbeta3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFbeta3 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 PDGFRalpha+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF) β super family. These molecules play important roles during many organogenic processes, even though they were originally identified as factors promoting the ectopic formation of cartilage and bone. The concentration of active BMP is controlled in part by inhibitors from three BMP binding protein families: short gastrulation/chordin, noggin and cerberus (reviewed by Balemans and Van Hul, 2002). Among these, chordin and noggin were the first proteins found to inhibit the activity of bound BMPs by preventing interactions with their BMP receptors (Piccolo et al., 1996; Zimmerman et al., 1996). Although noggin is encoded by a single gene in mammals, chordin belongs to a family of proteins that share a cysteine-rich pro-collagen repeat [or chordin-like cysteine-rich repeat (CR)], which is also found in various extracellular matrix proteins (reviewed by Garcia Abreu et al., 2002). Without exception, the homology between chordin family members lies within their CRs. The chordin polypeptide contains four CRs, of which the first and the third (CR1 and CR3) are responsible for BMP binding (Larrain et al., 2000). Binding of chordin to BMP4 is specific and tight (Piccolo et al., 1996). Proteolysis by Tolloid (or BMP1), which liberates CR1 and CR4 from chordin, is required to release bound BMP4 (Piccolo et al., 1997; Scott et al., 1999). The importance of CR for BMP interactions has been strengthened by the recent finding that connective tissue growth factor functions as a BMP-binding inhibitor, and that its single CR domain is essential for this activity (Abreu et al., 2002). We previously described a small chordin-like secreted protein, CHL1 (for chordin-like 1, re-designated from CHL), a novel BMP-binding inhibitor with three CRs (Nakayama et al., 2001). CHL1 was isolated originally from mouse bone marrow stromal cells. Interestingly, CHL1 expression 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.

OBJECTIVETo assess the capacities of the cytokine inhibitors interleukin-1 receptor antagonist (IL-1Ra; anakinra) and PEGylated soluble tumor necrosis factor receptor I (PEG sTNFRI; pegsunercept) to suppress neovascularization.METHODSA corneal angiogenesis assay was performed by implanting nylon discs impregnated with an angiogenic stimulator (basic fibroblast growth factor or vascular endothelial growth factor) into one cornea of female Sprague-Dawley rats. Animals were treated with IL-1Ra or PEG sTNFRI for 7 days, after which new vessels were quantified. In a parallel study, male Lewis rats with mycobacteria-induced adjuvant-induced arthritis were treated with IL-1Ra or PEG sTNFRI for 7 days beginning at disease onset, after which scores for inflammation and bone erosion as well as capillary counts were acquired from sections of arthritic hind paws.RESULTSTreatment with IL-1Ra yielded a dose-dependent reduction in growth factor-induced corneal angiogenesis, while PEG sTNFRI did not. IL-1Ra, but not PEG sTNFRI, significantly reduced the number of capillaries in arthritic paws, even though both anticytokines reduced inflammation and bone erosion to a similar degree.CONCLUSIONThese data support a major role for IL-1, but not TNFalpha, in angiogenesis and suggest that an additional antiarthritic mechanism afforded by IL-1 inhibitors, but not anti-TNF agents, is the suppression of the angiogenic component of pannus.

BackgroundAdjuvant 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.2ObjectivesThe current study was performed to assess whether anti-inflammatory molecules act to spare bone by regulating intra-lesional numbers of osteoclasts.MethodsAdA 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.ResultsDose-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.ConclusionThese data indicate that anti-inflammatory doses of IL-1ra and PEG sTNF-RI protect the structural integrity of arthritic joints by regulating osteoclast numbers.ReferencesKong, et al. Nature 1999;402:304 Feige, et al. Cell Mol Life Sci. 2000;57:1457

Background Marked polyarthritis is induced in male Lewis rats by injection of either heat-killed Mycobacterium tuberculosis in mineral oil (AdA) or lipoidal amine in complete Freund’s adjuvant (LA/CFA). For both models, paw swelling and body weight loss occur in all rats by 10 to 12 days after immunisation. Objectives Interestingly, however, the present study demonstrates that the morphologic and densitometric changes of AdA and LA/CFA are distinguishable. Methods We induced AdA or LA/CFA in male Lewis rats (180 g) and evaluated both clinical (body and spleen weight, volume of the tibiotarsal [hock] joints) and morphologic changes (bone mineral density [BMD] by DEXA; histopathologic lesions in the hock and femorotibial [knee] joints). Readings were obtained at the day of onset (as determined by initial paw swelling) as well as 5, 10 and 25 days after onset. Results Paw swelling, loss of body weight, enhanced spleen weight were identical in AdA and LA/CFA. In contrast, hock inflammation score, hock erosion score and hock cartilage score were lower in LA/CFA, though not statistically significant from AdA. Lesions developed more slowly in LA/CFA than in AdA. However, hock BMD as well as knee inflammation and erosion scores found to be different (shown here at Onset + 5 days): Conclusion Inflammatory and skeletal changes for AdA and LA/CFA were similar by clinical and histopathologic measures 10 days after onset, while inflammation receded by Onset + 25 days in LA/CFA more than it did in AdA. These data indicate that bone densitometric and morphologic findings can distinguish these two models of adjuvant arthritis despite their similar clinical presentations. This finding may be relevant in screening novel therapeutic candidates as these models may define different potential efficacious effects of a compound.