Faculty Bibliography

Murine macrophages are activated by interferon-gamma (IFN-gamma) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-alpha (IL-4Ralpha)-dependent manner. Macrophages activated in this way are designated as "alternatively activated" (M2a) macrophages. We have shown previously that adenosine A2A receptor (A2AR) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an "M2-like" phenotype that we have termed "M2d." Adenosine signaling suppresses the TLR-dependent expression of TNF-alpha, IL-12, IFN-gamma, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Ralpha gene (IL-4Ralpha(-/-) mice) that this adenosine-mediated switch does not require IL-4Ralpha-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-alpha and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-alpha), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify "M2" macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

BACKGROUND AND PURPOSE: We previously reported that adenosine, acting at adenosine A2A receptors (A2A R), inhibits osteoclast (OC) differentiation in vitro (A2A R activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2A R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2A R signalling. EXPERIMENTAL APPROACH: OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2A R agonist CGS21680, the A2A R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. KEY RESULTS: A2A R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2A R stimulation inhibited p50/p105 NFkappaB nuclear translocation in control but not in PKA KO cells. A2A R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2A R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. CONCLUSIONS AND IMPLICATIONS: A2A R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFkappaB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.

Bone is a dynamic organ that undergoes continuous remodeling while maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis. In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G protein-coupled receptors, and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as in osteoblast differentiation and bone formation. Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis. These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology.

OBJECTIVES: We and others have previously demonstrated that methotrexate (MTX) mediates its anti-inflammatory effects through an increase in cellular release of adenosine. Consistent with this observation, there is no increase in adenosine from exudates of mouse strains resistant to MTX. Because intracellular MTX polyglutamates inhibit AICAR transformylase (ATIC) activity and thereby promote adenosine release we determined whether there is any difference in the capacity of cells from MTX-resistant mice to accumulate MTX polyglutamates. METHODS: Dermal fibroblasts (DF) from BALBc, MTX-sensitive, and DBA/1J, MTX-resistant, mice were cultured in the presence or absence of MTX. Adenosine concentration in the supernatant and intracellular MTX polyglutamate (MTXPG1-5) concentrations were measured by liquid chromatography. ATIC activity in DF was monitored spectrophotometrically by the formation of formytetrahydrofolate. RESULTS: MTX (1 muM) increased adenosine production by DF from BALBc sensitive-mice from 269+/-40 nM to 446+/-4 nM. No adenosine production was found in supernates of cultured DF from DBA/1J mice regardless of MTX treatment. Intracellular MTX polyglutamates (MTXPG2-4) were detected only in BALBc DFs, not in DBA/1J DF. Further investigation demonstrated that ATIC activity was inhibited following MTX treatment in DF from BALBc mice. CONCLUSIONS: These data suggest that resistance to the anti-inflammatory effects of MTX could be due to diminished MTX polyglutamate accumulation resulting in diminished ATIC inhibition and adenosine accumulation.

OBJECTIVES: To evaluate the changes in types of medications prescribed for pain before and after withdrawal of certain selective cyclooxygenase 2 (COX-2) inhibitors in 2004 and to determine whether there was an association with fall events in elderly adults with a diagnosis of osteoarthritis (OA). DESIGN: A nested case-control design using electronic medical records compiled between 2001 and 2009. SETTING: Electronic medical records for care provided in an integrated health system in rural Pennsylvania over a 9-year period (2001-09), the midpoint of which rofecoxib and valdecoxib were pulled from the market. PARTICIPANTS: Thirteen thousand three hundred fifty-four individuals aged 65 to 89 with a diagnosis of OA. MEASUREMENTS: The incidence of falls and fractures was examined in relation to analgesics prescribed: narcotics, COX-2 inhibitors, and nonsteroidal anti-inflammatory drugs (NSAIDs). The comparison sample of individuals who did not fall was matched 3:1 with those who fell according to age, sex, and comorbidity. RESULTS: Narcotic analgesic prescriptions were associated with a significantly greater risk of falls and fractures. The likelihood of experiencing a fall/fracture was higher in participants prescribed narcotic analgesics than those prescribed a COX-2 inhibitor (odds ratio (OR) = 3.3, 95% confidence interval (CI) = 2.5-4.3) or NSAID (OR = 4.1, 95% CI = 3.7-4.5). CONCLUSION: Use of narcotic analgesics is associated with risk of falls and fractures in elderly adults with OA, an observation that suggests that the current guidelines for the treatment of pain, which include first-line prescription of narcotics, should be reevaluated.

Morning stiffness and increased symptoms of inflammatory arthritis are among the most common manifestations of rheumatoid arthritis (RA). Tumor necrosis alpha (TNF-alpha), an important mediator of inflammation in RA, regulates the circadian expression of clock proteins, and adenosine A(2A) receptors (A(2A)R) mediate many of the anti-inflammatory and antirheumatic actions of methotrexate, the cornerstone drug in the treatment of RA. We found that A(2A)R activation and TNF-alpha activated the clock core loop of the human monocytic THP-1 cell line. We further observed that interleukin (IL)-10, but not IL-12, mRNA expression fluctuates in a circadian fashion and that TNF-alpha and A(2A)R stimulation combined increased IL-10 expression. Interestingly, TNF-alpha, but not CGS21680, dramatically inhibited IL-12 mRNA expression. The demonstration that A(2A)R and TNF-alpha regulate the intrinsic circadian clock in immune cells provides an explanation for both the pathologic changes in circadian rhythms in RA and for the adverse circadian effects of methotrexate, such as fatigue.

Netrins have been extensively studied for their role in axonal guidance during neural development. In addition, netrins are chemopulsants for a variety of non-neuronal cell types via binding to their receptors Unc5b and DCC. Although thought to suppress inflammation in several settings, netrin1, acting via Unc5b, inhibits macrophage migration directed by chemokines CCL2 and CCL19 to promote macrophage retention in and exacerbation of atherosclerotic plaque. We asked whether Netrin1 was expressed during osteoclast (OC) differentiation and whether it plays a role in OC differentiation.DXAscan and MicroCT analysis were performed on Netrin1 deficient mice (radiation chimeras) and wildtype (WT, radiation chimeras) littermates. OC differentiation was studied as M-CSF/RANKL-stimulated differentiation of murine bone marrow precursors to TRAP+/multinucleated cells, in the presence/absence of recombinant Netrin1 and Unc5b antibody. Netrin1, Unc5b and DCC expression were studied by RT-PCR and Western Blot in primary bone marrow-derived osteoclasts. Netrin1 immunostaining was performed in human tissue obtained following primary prosthesis implantation or after prosthesis revision. During OC differentiation cell-associated Netrin1 and Unc5b (but not DCC) protein expression increased by 30+2% and 98+4% respectively (p<0.001,n=4) and Netrin1 secretion increased by 66+2% (p<0.001,n=4). Consistently, RANKL stimulates an increase in Netrin1 and Unc5b mRNA expression during OC differentiation (25+4 and 3+0.5 fold change respectively p<0.001,n=4). Moreover, in Netrin1-deficient marrow precursors OC differentiation was diminished by 65+2% as compared to control (p<0.001,n=6), an effect reversed by addition of recombinant netrin1 to cultures (121+5% increased, p<0.5,n=4). An antibody to the netrin1 receptor Unc5b reduces OC formation by 57+6% (p<0.001, =6) whereas an antibody to DCC had no effect on OC formation (5+4% reduction, p=NS vs. control,n=6). Finally, DXAscan and MicroCT analysis demonstrated an increase in!

ABSTRACT: It has been recently demonstrated that interleukin 1beta (IL-1beta) plays a central role in monosodium urate crystal-induced inflammation and that the NALP3 inflammasome plays a major role in IL-1beta production. These discoveries have offered new insights into the pathogenesis of acute gouty arthritis. In this review, we discuss the molecular mechanisms by which monosodium urate crystals induce acute inflammation and examine the mechanisms of action (MOAs) of traditional anti-inflammatory drugs (e.g., nonsteroidal anti-inflammatory drugs, colchicine, and glucocorticoids) and biologic agents (e.g., the IL-1beta antagonists anakinra, rilonacept, and canakinumab) to understand how their MOAs contribute to their safety profiles. Traditional anti-inflammatory agents may act on the IL-1beta pathway at some level; however, their MOAs are broad-ranging, unspecific, and biologically complex. This lack of specificity may explain the range of systemic adverse effects associated with them. The therapeutic margins of nonsteroidal anti-inflammatory drugs, colchicine, and glucocorticoids are particularly low in elderly patients and in patients with cardiovascular, metabolic, or renal comorbidities that are frequently associated with gouty arthritis. In contrast, the IL-1beta antagonists act on very specific targets of inflammation, which may decrease the potential for systemic adverse effects, although infrequent but serious adverse events (including infection and administration reactions) have been reported. Because these IL-1beta antagonists target an early event immediately downstream from NALP3 inflammasome activation, they may provide effective alternatives to traditional agents with minimal systemic adverse effects. Results of ongoing trials of IL-1beta antagonists will likely provide clarification of their potential role in the management of acute gouty arthritis.

INTRODUCTION: Adenosine, acting through the A2A receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A2A receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A2A receptor produces its fibrogenic effects. METHODS: The effects of A2A receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A2A receptor stimulation on CTGF and TGF-beta1 expression are also examined. RESULTS: A2A receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A2A receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A2A receptor-mediated enhancement of collagen type I production. A2AR activation, however, resulted in a decrease in TGF-beta1 protein release. CONCLUSIONS: Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A2A receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.