Adenosine inhibition of lipopolysaccharide-induced interleukin-6 secretion by the osteoblastic cell line MG-63.

Adenosine is known to inhibit inflammatory responses in many cell systems via a family of purine receptors termed "P1." The P1 family consists of the adenosine receptors (ADORA) of subtypes A(1), A(2a), A(2b), and A(3). In order to assess whether adenosine has anti-inflammatory actions in osteoblastic cells, we investigated its effects on lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release in an in vitro inflammatory functional response model. We showed that the osteoblastic cell line MG-63 expresses ADORA(1), A(2a), and A(2b) but not A(3). Treatment of MG-63 cells with adenosine and pharmacological ADORA agonist 5'-N-ethylcarboxamidoadenosine or 2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) inhibits LPS-induced IL-6 release. This inhibition was protein kinase A (PKA)-dependent and mimicked by treatment with the adenylate cyclase activator forskolin. Treatment of MG-63 with the ADORA(2a)-specific antagonist ZM241385 partially reversed the inhibitory effects of ADORA stimulation on LPS-induced IL-6 release. Overall, these data suggest that ADORA(2a) is involved in the regulation of LPS-induced IL-6 release, thus illustrating a regulatory role for adenosine receptors in the control of inflammation and potentially osteoclastogenesis and bone resorption.

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Inhibition of adenosine kinase attenuates interleukin-1- and lipopolysaccharide-induced alterations in articular cartilage metabolism.

OBJECTIVE: To investigate the effect of adenosine kinase inhibition on interleukin (IL)-1beta- and lipopolysaccharide (LPS)-induced cartilage damage. DESIGN: Articular cartilage was obtained from the metacarpophalangeal joints of 10 young adult horses. Following a stabilization period, weighed cartilage explants were exposed to IL-1beta (10 ng/ml) or LPS (50 microg/ml) to induce cartilage degradation. To test the potential protective effects of adenosine, these explants were simultaneously exposed to adenosine (100 microM), the adenosine kinase inhibitor 5'iodotubercidin (ITU, 1 microM) or to both adenosine and ITU. After 72 h in culture, conditioned medium was collected for evaluation of glycosaminoglycan (GAG), nitric oxide (NO), prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP)-3 release. RESULTS: IL-1beta and LPS stimulated significant release of GAG, NO, PGE2 and MMP-3. Incubation with ITU significantly inhibited both IL-1beta- and LPS-induced GAG release, but did not alter MMP-3 production. Exposure to ITU also reduced IL-1beta-induced PGE2 release and LPS-induced NO production. Direct adenosine supplementation did not attenuate the effects of IL-1beta or LPS, and the addition of adenosine or ITU in the absence of IL-1beta or LPS did not have any detectable effect on cartilage metabolism in this model. CONCLUSIONS: The adenosine kinase inhibitor ITU attenuated experimentally induced cartilage damage in an in vitro cartilage explant model. Release of adenosine from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions and modulation of these pathways in the joint may have potential for treatment of arthropathies.

Effects of an adenosine kinase inhibitor and an adenosine deaminase inhibitor on accumulation of extracellular adenosine by equine articular chondrocytes.

OBJECTIVE: To investigate accumulation of extracellular adenosine (ADO) by equine articular chondrocytes and to compare effects of adenosine kinase inhibition and adenosine deaminase inhibition on the amount of nitric oxide (NO) produced by lipopolysaccharide (LPS)-stimulated chondrocytes. SAMPLE POPULATION: Articular cartilage from metacarpophalangeal and metatarsophalangeal joints of 14 horses. PROCEDURE: Chondrocytes were cultured as monolayers, and cells were incubated with LPS, the adenosine kinase inhibitor 5'-iodotubercidin (ITU), or the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA). Concentrations of ADO in cell supernatants were measured by use of reverse-phase high-performance liquid chromatography. Effect of inhibition of enzymatic metabolism of ADO on induced NO production was evaluated by exposing cells to a combination of LPS and ITU or LPS and EHNA. RESULTS: Articular chondrocytes accumulated extracellular ADO when exposed to LPS or ITU. Chondrocytes exposed to ITU accumulated ADO in a time-dependent manner. Unstimulated chondrocytes did not accumulate ADO. Similarly, EHNA alone did not produce detectable ADO concentrations; however, addition of EHNA and ITU resulted in a synergistic effect on accumulation of ADO. Lipopolysaccharide-induced NO production was more effectively suppressed by exposure to ITU than to EHNA CONCLUSIONS AND CLINICAL RELEVANCE: Equine articular chondrocytes release ADO in response to the proinflammatory stimulus of bacterial LPS. Inhibition of the metabolism of ADO increases accumulation of extracellular ADO. Autocrine release of ADO from chondrocytes may play a role in the cellular response to tissue damage in arthritic conditions, and pharmacologic modulation of these pathways in joints of arthritic horses could be a potential method of therapy.

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes.

OBJECTIVE: To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. PROCEDURE: Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. RESULTS: Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. CONCLUSIONS AND CLINICAL RELEVANCE: Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.