Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis.

BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Transglutaminase 2 is a marker of chondrocyte hypertrophy and osteoarthritis severity in the Hartley guinea pig model of knee OA.

OBJECTIVE: The transglutaminase (TG) isoenzyme TG2, which catalyzes protein cross-linking via transamidation, influences healing phenotype in multiple forms of tissue injury. Moreover, TG2 knockout suppresses cartilage destruction but promotes osteophyte formation in instability-induced mouse knee osteoarthritis (OA). TG2 is marker of growth plate chondrocyte hypertrophy. Moreover, TG2 secreted by chondrocytes acts in part by promoting chondrocyte maturation to hypertrophy, a differentiation state linked with MMP-13 expression and disease progression in OA. Moreover, glucosamine, which is currently under investigation as an OA therapy, binds and inhibits TG2. Here, we examined TG2 as a potential marker of cartilage hypertrophy in the spontaneous guinea pig model of OA. METHODS: Synovial fluid ELISA and cartilage Immunohistochemistry and quantitative Reverse transcription-polymerase chain reaction (RT-PCR), were used to examine TG2 expression and TG transamidation-catalyzed isopeptide bonds. RESULTS: TG isopeptide bonds and TG2 were most abundant in articular cartilage in early knee OA. TG2 expression was robust at sites of early but not established osteophytes. Synovial fluid TG2 correlated with knee OA total histological score (r=0.47, P=0.01), as did medial tibial plateau cartilage TG2 mRNA (r=1.0, P=0.003). At 12 months of age, medial tibial plateau cartilage TG2 mRNA expression rose markedly in association with elevated type X collagen, as well as ADAMTS-5, and MMP-13 expression, changes not shared in age-matched Strain 13 guinea pigs that are less susceptible to knee OA. CONCLUSION: Hartley guinea pig knee TG2 expression associates with enhanced articular chondrocyte hypertrophy and is a biomarker of OA severity.

Calcification of human articular knee cartilage is primarily an effect of aging rather than osteoarthritis.

OBJECTIVE: Pathologic calcification of articular cartilage in human knees is often associated with advanced age and conditions of osteoarthritis (OA). Coincidently, most studies that have characterized calcification in joint cartilage have examined populations that are aged and presenting with clinical symptoms. Generally, these studies rely upon relatively insensitive plain radiographs or synovial fluid crystal analyses to quantify calcium levels. The purpose of this study was to examine the relationship between cartilage calcification and aging in an unselected donor population of diverse age using highly sensitive calcification imaging. METHODS: A group of 106 knee blocks were obtained from 56 individual donors (25 females and 31 males, aged 12-74, avg. 50.3 years). Condylar surfaces were graded on a 4-point OA grading scale for cartilage degeneration. The condyles were cut into approximately 7-10mm thick slabs. Using a Faxitron radiography system, high-resolution images were taken of the slabs to specifically image calcification in cartilage. The quantified calcification areas were then analyzed and correlations with both OA grade and age were assessed. RESULTS: Every knee presented some measurable calcification. The relative calcium deposition had a significant positive correlation with age. This same positive correlation was seen between condyles showing grade 1 and 2 changes. OA grades higher than 2 did not present any further significant increase in calcium levels. CONCLUSION: These observations indicate that age rather than OA is the predominant factor driving progressive pathologic calcification in articular cartilage.

Inorganic pyrophosphate generation and disposition in pathophysiology.

Inorganic pyrophosphate (PP(i)) regulates certain intracellular functions and extracellular crystal deposition. PP(i) is produced, degraded, and transported by specialized mechanisms. Moreover, dysregulated cellular PP(i) production, degradation, and transport all have been associated with disease, and PP(i) appears to directly mediate specific disease manifestations. In addition, natural and synthetic analogs of PP(i) are in use or currently under evaluation as prophylactic agents or therapies for disease. This review summarizes recent developments in the understanding of how PP(i) is made and disposed of by cells and assesses the body of evidence for potentially significant physiological functions of intracellular PP(i) in higher organisms. Major topics addressed are recent lines of molecular evidence that directly link decreased and increased extracellular PP(i) levels with diseases in which connective tissue matrix calcification is disordered. To illustrate in depth the effects of disordered PP(i) metabolism, this review weighs the roles in matrix calcification of the transmembrane protein ANK, which regulates intracellular to extracellular movement of PP(i), and the PP(i)-generating phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1).

Lyme disease 2000. Emerging zoonoses complicate patient work-up and treatment.

Lyme disease and other tick-borne diseases affect all age groups, including active persons age 50 and older. There has been a steady expansion in case numbers and the geographic borders of Lyme disease over the last two decades. Better recognition of two emerging tick-borne zoonoses (babesiosis and human granulocytic ehrlichiosis) that can be co-transmitted with Lyme disease is changing the approach to diagnosis and treatment of Lyme disease. Advances from genetic cloning have improved our understanding of the immune mechanisms associated with treatment-resistant Lyme arthritis and enabled the development of the first Lyme disease vaccine. The available vaccine is less efficacious in persons age 65 and older than in younger adults, but it may be a useful adjunct to other preventive measures in areas endemic for Lyme disease.

Interleukin-8 and its receptor CXCR2 in atherosclerosis.

The participation of inflammatory cells in atherosclerosis is a well-known process that involves numerous molecules including chemotactic cytokines (chemokines) for their entry into the vessel wall. Although the C-C chemokine monocyte chemoattractant protein-1 and its receptor, CCR2, have been implicated in atherosclerosis, the role of the classic C-X-C chemokine, interleukin-8 (KC/growth-related oncogene alpha in mice) and its receptor CXCR2 has not been studied in the pathogenesis of atherosclerosis. Our research has shown that CXCR2 is strongly expressed on macrophages (Mphi) in atherosclerotic lesion. This CXCR2 expression is proatherogenic in that CXCR2 deficiency significantly reduces the progression of advanced atherosclerosis in mice. Although the mechanism still needs to be worked out, it appears that CXCR2 expression on lesion Mphi is essential for these cells to be retained in the lesion.

Noninvasive evaluation of adult onset myopathy from carnitine palmitoyl transferase II deficiency using proton magnetic resonance spectroscopy.

OBJECTIVE: The adult onset metabolic myopathy of carnitine palmitoyl transferase II (CPT II) deficiency is under-recognized, in part due to variable degrees of enzyme deficiency and symptomatology, as well as limitations in means for noninvasive evaluation. We describe a proton magnetic resonance spectroscopy (MRS) technique, using a standard clinical magnetic resonance imaging scanner, to diagnose and help monitor the response to therapy in adult CPT II deficiency. METHODS: A 53-year-old woman presented with a long standing history of diffuse aching and fatigue provoked by high fat intake, fasting, or prolonged exertion. Muscle biopsy revealed myopathic features and a deficiency (33% of control) of CPT II activity with elevated palmitoyl carnitine. Proton MRS of the soleus muscle was performed using a 1.5 Tesla scanner before and during dietary therapy. RESULTS: Proton MRS revealed shortening of the transverse relaxation time (T2), consistent with increased acetylation of the carnitine pool. The symptoms resolved completely by treatment with frequent feedings of a high carbohydrate diet low in long chain fatty acids supplemented with medium chain triglycerides and L-carnitine. Recovery of normal muscle MRS and carnitine T2 relaxation was documented by the third month of therapy. CONCLUSION: Proton MRS is a novel, potentially useful, and readily available adjunct in the diagnosis and therapeutic monitoring of muscle CPT II deficiency.

Clinical trials review: crystal deposition diseases.

This review focuses on several recent clinically-related observations concerning Calcium Pyrophosphate Deposition Disease (CPPDD). Developments include new information on the epidemiology of idiopathic CPPDD in aging, on familial CPPDD, and on associated conditions that predispose to secondary CPPDD. Factors that may precipitate pseudogout, including the use of intra-articular hyaluronan and systemic G-CSF, are also discussed.

New developments in the pathogenesis of articular cartilage calcification.

Articular cartilage, unlike growth plate cartilage, is specialized to not undergo matrix calcification. However, articular cartilage mineralization, in the form of CPPD (chondrocalcinosis) and hydroxyapatite crystals, frequently accompanies and complicates osteoarthritis and aging. Recent work has demonstrated that certain features of growth cartilage development and mineralization are shared in degenerative cartilage. These include chondrocyte proliferation, hypertrophy and increased apoptosis. Moreover, parathyroid hormone related protein (PTHrP), one of the central mediators of endochondral development, is abundant in osteoarthritic cartilage. Cartilage PPi elaboration and cytosolic transglutaminase activity are markedly increased with aging. Only recently have the molecular identities been defined for the chondrocyte inorganic pyrophosphate (PPi)-generating isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family (including PC-1 and B10), and for transglutaminase in articular cartilage. This review focuses on the evolving understanding of the potential roles, in articular cartilage calcification, of PTHrP, PDNP family enzymes, PPi metabolism, and transglutaminase activity.

Bone morphogenetic proteins and bFGF exert opposing regulatory effects on PTHrP expression and inorganic pyrophosphate elaboration in immortalized murine endochondral hypertrophic chondrocytes (MCT cells).

A fundamental question in endochondral development is why the expression of parathyroid hormone-related protein (PTHrP), which inhibits chondrocyte maturation and mineralization, becomes attenuated at the stage of chondrocyte hypertrophy. To address this question, we used clonal, phenotypically stable SV40-immortalized murine endochondral chondrocytes that express a growth-arrested hypertrophic phenotype in culture (MCT cells). Addition of individual cytokines to the medium of MCT cells revealed that bone morphogenetic protein (BMP)-6, which commits chondrocytes to hypertrophy, markedly inhibited PTHrP production. This activity was shared by three other osteogenic bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7) and by transforming growth factor beta (TGF-beta), which all inhibited the level of PTHrP mRNA. In contrast, basic fibroblast growth factor (bFGF), an inhibitor of chondrocyte maturation to hypertrophy, induced PTHrP in MCT cells and antagonized the effects of BMP-2, BMP-4, BMP-6, and BMP-7 and TGF-beta on PTHrP expression. Opposing effects of bFGF and BMPs also were exerted on the elaboration of inorganic pyrophosphatase (PPi), which regulates the ability of hypertrophic chondrocytes to mineralize the matrix. Specifically, BMP-2 and BMP-4, but not BMP-6 and BMP-7, shared the ability of TGF-beta to induce PPi release, and this activity was inhibited by bFGF in MCT cells. Our results suggest that effects on PTHrP expression could contribute to the ability of BMP-6 to promote chondrocyte maturation. BMPs and bFGF exert opposing effects on more than one function in immortalized hypertrophic chondrocytes. Thus, the normal decrease in bFGF responsiveness that accompanies chondrocyte hypertrophy may function in part by removing the potential for bFGF to induce PTHrP expression and to oppose the effects of BMPs. MCT cells may be useful in further understanding the mechanisms regulating the differentiation and function of hypertrophic chondrocytes.

A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.

Chronic macrophage-mediated inflammation is central to atherosclerosis. A role of the monocyte chemotactic and activating C-C chemokine JE/monocyte chemotactic protein-1 has been proposed. However, the human C-X-C chemokines growth-regulated oncogene (GROalpha) and IL-8, and their shared receptor, CXCR-2, also can be expressed at sites of chronic inflammation. Because we detected CXCR-2 in the intima of human atherosclerotic lesions, we examined the role of leukocyte CXCR-2 expression in affecting lesion cellularity. Atherosclerosis-susceptible LDL receptor-deficient mice were irradiated, successfully repopulated with bone marrow cells that either lacked or expressed mIL-8RH (the homologue of CXCR-2), and fed an atherogenic diet for 16 wk. In recipients of mIL-8RH+/+ marrow, mIL-8RH colocalized with densely accumulated intimal MOMA-2 positive macrophages. In contrast, lesions in recipients of mIL-8RH-/- marrow lacked mIL-8RH, had little intimal MOMA-2 staining, and were less extensive. The mIL-8RH ligand KC/GROalpha was detected in the intima of all aortic atherosclerotic lesions. Thus, the capacity of leukocytes to express mIL-8RH, and associated intralesional expression of its ligands such as KC/GROalpha, mediated the intimal accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice.

Hypercalcemia and systemic lupus erythematosus.

Hypercalcemia is commonly caused by the increased production of parathyroid hormone-related protein (PTHrP) by a malignancy. In fact, the demonstration of increased PTHrP production in a patient with hypercalcemia is virtually pathognomonic of malignancy. We studied a patient with systemic lupus erythematosus (SLE), generalized lymphadenopathy, and hypercalcemia. Immunohistology of 2 biopsied lymph nodes revealed the abundant expression of PTHrP and the absence of malignant transformation. Although apparently rare, PTHrP production by non-malignant lymphoid tissue may occur in SLE and should be considered in the differential diagnosis of hypercalcemia.

Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells.

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.

Oxidized LDL enhances lipopolysaccharide-induced tissue factor expression in human adherent monocytes.

Oxidized low-density lipoprotein (oxLDL) has been characterized as an atherogenic molecule responsible for the induction of a variety of gene products. One such gene, tissue factor (TF), the cellular initiator of the coagulation cascade, is not expressed in normal vascular tissue but is expressed by monocytes and foam cells in atherosclerotic lesions. Therefore, we examined the effect of oxLDL on TF expression in cultured human adherent monocytes. Endotoxin-free oxLDL alone did not induce TF expression in adherent monocytes. However, oxLDL significantly enhanced TF expression induced by the inflammatory mediator, bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In contrast, oxLDL did not alter LPS-mediated production of interleukin-8 and actually inhibited LPS-induced secretion of tumor necrosis factor-alpha, suggesting that some aspects of the signaling pathways for TF induction differ from those of other LPS-responsive monocyte/macrophage gene products. Thus, this study documents specific modulation of the expression of LPS-inducible genes in monocytic cells by oxLDL. Factors that enhance TF expression in monocyte/macrophage cells present in atheroma may contribute to the severity of thrombotic episodes and complications observed in atherosclerosis.

Gout and mechanisms of crystal-induced inflammation.

Since last year's review of gout and hyperuricemia, investigators have described new potential mechanisms that may contribute to urate crystal deposition and the propagation, self-limitation, and therapeutic control of gouty inflammation. The clinical presentation of gout in women continues to be described in greater detail. Also, new information on oral allopurinol desensitization is now available to help approach the difficult problem of allopurinol hypersensitivity.

Apolipoprotein (apo) E inhibits the capacity of monosodium urate crystals to stimulate neutrophils. Characterization of intraarticular apo E and demonstration of apo E binding to urate crystals in vivo.

Factors that modulate the ability of monosodium urate crystals to stimulate leukocytes could regulate gouty inflammation. Lipoproteins that bear apo B-100 and apo E bind to urate crystals and suppress crystal-neutrophil interaction. In this study, we observed that urate crystals, coated with apo E of monocyte origin, had a diminished ability to stimulate neutrophils. Apo E was also detected on the surface of urate crystals recovered from gout patients. Thus, we analyzed apo E in noninflammatory synovial fluid, and found it to be associated with particles of heterogeneous size and of predominantly alpha and pre-beta electrophoretic mobility. Local articular synthesis of at least a portion of synovial fluid apo E was suggested because (a) the synovial fluid/plasma concentration ratio of apo E was significantly higher than that for both apo B and apo A-I, which are not widely synthesized by extrahepatic tissues, (b) cultured rheumatoid synovial cells in first passage secreted apo E, (c) a portion of synovial fluid apo E was heavily sialylated. We conclude that synovial fluids contain apo E that appears partly of local origin. Apo E binds to urate crystals and could modulate gouty inflammation.