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1.  IgG galactosylation aberrancy precedes disease onset, correlates with disease activity and is prevalent in autoantibodies in rheumatoid arthritis 
Arthritis and rheumatism  2010;62(8):2239-2248.
To examine the association of IgG galactosylation aberrancy with disease parameters in rheumatoid arthritis (RA).
N-glycan analysis of serum from multiple cohorts was performed. IgG N-glycan content and timing of N-glycan aberrancy relative to disease onset was compared in healthy and RA subjects. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, gender, age, anti-CCP titer, disease duration, and CRP on aberrant galactosylation was determined using multivariate analysis. N-glycan content was also compared between epitope affinity purified autoantibodies and the remaining repertoire IgG in RA subjects.
Our results confirm the aberrant galactosylation of IgG in RA (1.36 ± 0.43) compared to healthy controls (1.01 ± 0.23) (P < 0.0001). We observe a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman rho = 0.37, p<0.0001). This correlation is higher in females [Spearman rho = 0.60 (P<0.0001)] than males [Spearman rho = 0.16 (P = 0.10)]. Further, IgG galactosylation aberrancy substantially predates onset of arthritis and the diagnosis of RA (3.5 years) and resides selectively in the anti-citrullinated peptide autoantibody fraction.
Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, that associates with disease activity in a gender specific manner, and that resides preferentially in autoantibodies.
PMCID: PMC4118465  PMID: 20506563
IgG Glycosylation; Gender Differences; Dysregulation; Humoral Immunity; Disease Onset; Autoantibody
2.  Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints 
PLoS ONE  2015;10(2):e0116237.
Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb’s binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.
PMCID: PMC4314068  PMID: 25642942
3.  Joint Tissues Amplify Inflammation and Alter Their Invasive Behavior via Leukotriene B4 in Experimental Inflammatory Arthritis 
Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB4 synthesis and to respond to LTB4 within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase–/– and leukotriene A4 (LTA4) hydrolase–/– mice, we demonstrate that FLSs generate sufficient levels of LTB4 production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs—which comprise the predominant lineage populating the synovial lining—are competent to metabolize exogenous LTA4 into LTB4 ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB4 3-fold without inducing the expression of LTA4 hydrolase protein. Moreover, LTB4 (acting via LTB4 receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB4 in joints, placing LTB4 regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.
PMCID: PMC3690310  PMID: 20876351
4.  Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis* 
Science translational medicine  2013;5(209):209ra150.
Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.
PMCID: PMC4032227  PMID: 24174326
5.  Proteomic analysis of synovial fluid from the osteoarthritic knee: comparison with transcriptome analyses of joint tissues 
Arthritis and rheumatism  2013;65(4):981-992.
The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. A proteomic analysis of knee SF from OA patients and control subjects was performed and compared to microarray expression data from cartilage and synovium.
Age-matched knee SF samples from control subjects, and patients with early- and late-stage OA (n=10 per group) were compared using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A MS with multiplexed peptide selected reaction monitoring (SRM) assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity pathway analysis and compared to published synovial tissue and cartilage mRNA profiles.
66 proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute phase response, and the complement and coagulation pathways. Differential expression of 5 proteins was confirmed by SRM assay. A focused analysis of transcripts corresponding to the differentially present proteins indicates that both synovial and cartilage tissues may contribute to the OA SF proteome.
Proteins involved in the acute phase response, complement and coagulation pathways are differentially regulated in SF of OA patients suggesting they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.
PMCID: PMC3618606  PMID: 23400684
6.  The CJIE1 prophage of Campylobacter jejuni affects protein expression in growth media with and without bile salts 
BMC Microbiology  2014;14:70.
The presence of Campylobacter jejuni temperate bacteriophages has increasingly been associated with specific biological effects. It has recently been demonstrated that the presence of the prophage CJIE1 is associated with increased adherence and invasion of C. jejuni isolates in cell culture assays.
Quantitative comparative proteomics experiments were undertaken using three closely related isolates with CJIE1 and one isolate without CJIE1 to determine whether there was a corresponding difference in protein expression levels. Initial experiments indicated that about 2% of the total proteins characterized were expressed at different levels in isolates with or without the prophage. Some of these proteins regulated by the presence of CJIE1 were associated with virulence or regulatory functions. Additional experiments were conducted using C. jejuni isolates with and without CJIE1 grown on four different media: Mueller Hinton (MH) media containing blood; MH media containing 0.1% sodium deoxycholate, which is thought to result in increased expression of virulence proteins; MH media containing 2.5% Oxgall; and MHwithout additives. These experiments provided further evidence that CJIE1 affected protein expression, including virulence-associated proteins. They also demonstrated a general bile response involving a majority of the proteome and clearly showed the induction of almost all proteins known to be involved with iron acquisition. The data have been deposited to the ProteomeXchange with identifiers PXD000798, PXD000799, PXD000800, and PXD000801.
The presence of the CJIE1 prophage was associated with differences in protein expression levels under different conditions. Further work is required to determine what genes are involved in causing this phenomenon.
PMCID: PMC4004267  PMID: 24641125
Campylobacter jejuni; Prophage; Proteomics; iTRAQ; Bile response; Iron acquisition
7.  Platelets Participate in Synovitis via Cox-1–Dependent Synthesis of Prostacyclin Independently of Microparticle Generation 
In addition to the well-described role of platelets in thrombosis, a growing body of evidence implicates platelets in diverse inflammatory responses. We recently showed platelets can contribute to the pathophysiology of inflammatory arthritis via IL-1–containing microparticles. In this study, we demonstrate that platelets, and not platelet microparticles, actively contribute to synovitis via production of proinflammatory prostacyclin in an autoimmune arthritis model. Using both genetic and pharmacologic approaches, we establish that paracrine production of prostacyclin proceeds in the absence of cyclooxygenase-2. Furthermore, we also demonstrate that prostacyclin generation can arise via transcellular collaboration between platelets and fibroblast-like synoviocytes. In addition to shedding light on an unappreciated pathway of lipid synthesis in arthritis, we further delineate a novel effector activity by which platelets can contribute to inflammatory disease.
PMCID: PMC3208424  PMID: 21357261
8.  C5a receptor enables participation of mast cells in immune complex arthritis independent of Fc γ receptor modulation 
Arthritis and rheumatism  2010;62(11):3322-3333.
Mast cells are tissue-resident immune sentinels implicated in the pathogenesis of inflammatory joint disease. We hypothesized that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.
In vivo studies employed murine K/BxN arthritis, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR−/− and control mast cells were engrafted into mast cell-deficient W/Wv mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR−/− animals and in murine and human mast cell cultures.
Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR−/− mast cells adoptively transferred into W/Wv mice were incompetent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate expression of Fc γ receptors (FcγR) or otherwise alter activation threshold. In human mast cells, C5a promoted production of the neutrophil chemotaxin interleukin 8, and recruitment of neutrophils at 24h after serum administration was impaired in C5aR−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.
Stimulation via C5aR is required to unleash the pro-inflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism distinct from C5a-modulated FcγR expression.
PMCID: PMC2970731  PMID: 20662064
9.  A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis 
EMBO molecular medicine  2010;2(5):172-187.
Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering 9 members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the proinflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.
PMCID: PMC3058614  PMID: 20432503
secreted phospholipase A2; arthritis; autoimmunity; inflammation
10.  Leukotriene B4 driven neutrophil recruitment to the skin is essential for allergic skin inflammation 
Immunity  2012;37(4):747-758.
Scratching triggers skin flares in atopic dermatitis (AD). We demonstrate that scratching of human skin, and tape stripping of mouse skin, causes neutrophil influx. This influx in mice was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1−/− mice, and required expression of BLT1 on both T cells and non-T cells. Co-transfer of WT neutrophils, but not neutrophils deficient in BLT1 or the LTB4 synthesizing enzyme LTA4H, restored the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.
PMCID: PMC3478399  PMID: 23063331
11.  Mouse Mast Cell Tryptase mMCP-6 Is a Critical Link between Adaptive and Innate Immunity in the Chronic Phase of Trichinella spiralis Infection1 
Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established, their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is associated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.
PMCID: PMC2969178  PMID: 18354212
12.  Cadherin-11 Promotes Invasive Behavior of Fibroblast-like Synoviocytes 
Arthritis and rheumatism  2009;60(5):1305-1310.
To define the expression pattern of cadherin-11 in destructive pannus tissue of patients with rheumatoid arthritis and to determine if cadherin-11 expression in fibroblast-like synoviocytes controls their invasive capacity.
Cadherin-11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin-11 in regulating the invasive behavior of fibroblast-like synoviocytes, we generated L-cell clones expressing wild-type cadherin-11, mutant cadherin-11, and empty vector transfected controls. The invasive capacity of L-cell transfectants and cultured fibroblast-like synoviocytes treated with a blocking cadherin-11-Fc protein or control immunoglobulin was determined in Matrigel invasion assays.
Immunohistochemistry revealed that cadherin-11 is abundantly expressed in cells at the cartilage-pannus junction in rheumatoid synovitis. Invasion assays demonstrate a twofold increased invasive capacity of cadherin-11 transfected L-cells compared to L-cells transfected with E-cadherin or control vector. The invasive behavior of the L-cells stably transfected with a cadherin-11 construct that lacked the juxta-membrane cytoplasmic domain (cadherin-11 ΔJMD) was diminished to the level of vector control L-cells. Further, treatment with the cadherin-11-Fc fusion protein diminished the invasive capacity of fibroblast-like synoviocytes.
These in vitro studies implicate a role for cadherin-11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast-like synoviocytes in chronic synovitis and rheumatoid arthritis.
PMCID: PMC3764540  PMID: 19404963
Cadherin-11; Fibroblast-like Synoviocytes; Cell Invasion
13.  Thrombin-activatable carboxypeptidase B-cleavage of osteopontin regulates neutrophil survival and synoviocyte binding in rheumatoid arthritis 
Arthritis and rheumatism  2009;60(10):2902-2912.
Osteopontin (OPN) is a pro-inflammatory cytokine important in rheumatoid arthritis (RA). OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal α4β1 and α9β1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also termed thrombin-activatable fibrinolysis inhibitor (TAFI), removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell binding. We investigated the roles of OPN-R and OPN-L in: (i) synoviocyte adhesion, which contributes to formation of invasive pannus, and (ii) neutrophil survival, which affects inflammatory infiltrates, in RA.
Methods and Results
We developed ELISAs specific for OPN-R and OPN-L, and demonstrate elevations of OPN-R and OPN-L in RA, but not in osteoarthritis or psoriatic arthritis, synovial fluid samples. OPN-R and OPN-L levels correlated with multiple inflammatory cytokines including TNFα and IL-6. Immunohistochemical analyses demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an anti-apoptotic effect on neutrophils. OPN-R, but not OPN-L, augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to α4β1.
Thrombin activation of OPN (OPN-R) and its subsequent inactivation by thrombin-activatable CPB (OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA, by regulating neutrophil viability and reducing synoviocyte adhesion.
PMCID: PMC3757557  PMID: 19790060
osteopontin; thrombin-activatable carboxypeptidase B; thrombin-activatable fibrinolysis inhibitor; rheumatoid arthritis
14.  Murine CD7 Shares Antigenic Cross-Reactivity with HSP-60 
Hybridoma (2005)  2008;27(2):81-89.
Human (h) CD7 is a 40 kDa single chain Ig superfamily molecule that is expressed on thymocytes, a major subunit of peripheral T cells, and most natural killer cells. Ligands for hCD7 include the epithelial cell-produced molecule, K-12, and galectin. Mice deficient in CD7 have been shown to be resistant to LPS-induced endotoxic shock syndromes. However, monoclonal antibodies (MAb) to mouse (m) CD7 have yet to be produced, nor is the distribution of mCD7 protein in mice known. We have raised a panel of three rat MAbs to mCD7 by immunizing rats with recombinant mCD7 protein. However, using Western blot and immunoprecipitation of tissue extracts from mouse thymus, spleen, liver, brain, lymph node and skin, these anti-mouse CD7 MAbs bound only to murine heat shock protein 60 (HSP-60) present both in wild-type (CD7+/+) and CD7-deficient (CD7−/−) mice. Epitope mapping of the sites on HSP-60 and recombinant mCD7 recognized by mCD7 MAbs demonstrated non-homologous amino acid sequence epitopes recognized by anti-CD7 MAbs on both proteins. These data demonstrated molecular mimicry of mCD7 with HSP-60, and leave open the question of surface expression of mCD7.
PMCID: PMC3652665  PMID: 18642672
15.  Comparisons of Immunoassay and Mass Spectrometry Measurements of Serum Estradiol Levels and Their Influence on Clinical Association Studies in Men 
Immunoassay-based techniques, routinely used to measure serum estradiol (E2), are known to have reduced specificity, especially at lower concentrations, when compared with the gold standard technique of mass spectrometry (MS). Different measurement techniques may be responsible for the conflicting results of associations between serum E2 and clinical phenotypes in men.
Our objective was to compare immunoassay and MS measurements of E2 levels in men and evaluate associations with clinical phenotypes.
Design and Setting:
Middle-aged and older male subjects participating in the population-based Osteoporotic Fractures in Men (MrOS) Sweden study (n = 2599), MrOS US (n = 688), and the European Male Aging Study (n = 2908) were included.
Main Outcome Measures:
Immunoassay and MS measurements of serum E2 were compared and related to bone mineral density (BMD; measured by dual energy x-ray absorptiometry) and ankle-brachial index.
Within each cohort, serum E2 levels obtained by immunoassay and MS correlated moderately (Spearman rank correlation coefficient rS 0.53–0.76). Serum C-reactive protein (CRP) levels associated significantly (albeit to a low extent, rS = 0.29) with immunoassay E2 but not with MS E2 levels. Similar associations of immunoassay E2 and MS E2 were seen with lumbar spine and total hip BMD, independent of serum CRP. However, immunoassay E2, but not MS E2, associated inversely with ankle-brachial index, and this correlation was lost after adjustment for CRP.
Our findings suggest interference in the immunoassay E2 analyses, possibly by CRP or a CRP-associated factor. Although associations with BMD remain unaffected, this might imply for a reevaluation of previous association studies between immunoassay E2 levels and inflammation-related outcomes.
PMCID: PMC3667264  PMID: 23633197
16.  The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: the microparticle-associated immune complexes 
EMBO Molecular Medicine  2012;5(2):235-249.
Immunoglobulins, antigens and complement can assemble to form immune complexes (IC). ICs can be detrimental as they propagate inflammation in autoimmune diseases. Like ICs, submicron extracellular vesicles termed microparticles (MP) are present in the synovial fluid from patients affected with autoimmune arthritis. We examined MPs in rheumatoid arthritis (RA) using high sensitivity flow cytometry and electron microscopy. We find that the MPs in RA synovial fluid are highly heterogeneous in size. The observed larger MPs were in fact MP-containing ICs (mpICs) and account for the majority of the detectable ICs. These mpICs frequently express the integrin CD41, consistent with platelet origin. Despite expression of the Fc receptor FcγRIIa by platelet-derived MPs, we find that the mpICs form independently of this receptor. Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti-citrullinated peptide antibodies contributes to the production of mpICs. Functionally, platelet mpICs are highly pro-inflammatory, eliciting leukotriene production by neutrophils. Taken together, our data suggest a unique role for platelet MPs as autoantigen-expressing elements capable of perpetuating formation of inflammatory ICs.
PMCID: PMC3569640  PMID: 23165896
arthritis; autoantigens; immune complexes; microparticles; platelets
17.  Synergistic inhibition of mesothelioma cell growth by the combination of clofarabine and resveratrol involves Nrf2 downregulation 
BMB Reports  2012;45(11):647-652.
We previously reported that MSTO-211H cells have a higher capacity to regulate Nrf2 activation in response to changes in the cellular redox environment. To further characterize its biological significance, the response of Nrf2, a transcription factor that regulates ARE-containing genes, on the synergistic cytotoxic effect of clofarabine and resveratrol was investigated in mesothelioma cells. The combination treatment showed a marked growth-inhibitory effect, which was accompanied by suppression of Nrf2 activation and decreased expression of heme oxygenase-1 (HO-1). While transient overexpression of Nrf2 conferred protection against the cytotoxicity caused by their combination, knockdown of Nrf2 expression using siRNA enhanced their cytotoxic effect. Pretreatment with Ly294002, a PI3K inhibitor, augmented the decrease in HO-1 level by their combination, whereas no obvious changes were observed in Nrf2 levels. Altogether, these results suggest that the synergistic cytotoxic effect of clofarabine and resveratrol was mediated, at least in part, through suppression of Nrf2 signaling. [BMB Reports 2012; 45(11): 647-652]
PMCID: PMC4133806  PMID: 23187004
Chemoresistance; Clofarabine; Mesothelioma; Nrf2; Resveratrol
18.  Neutrophil-derived leukotriene B4 is required for inflammatory arthritis 
Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
PMCID: PMC2118292  PMID: 16567388
19.  Identification of a central role for complement in osteoarthritis 
Nature Medicine  2011;17(12):1674-1679.
Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.
PMCID: PMC3257059  PMID: 22057346
20.  Mast cells contribute to autoimmune inflammatory arthritis via their tryptase•heparin complexes1 
Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis (RA), MC’s contribution to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase•heparin complexes have pro-inflammatory activity, and significant amounts of hTryptase-β are present in RA synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase•heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/B×N mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-β•heparin or mMCP-6•heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase•heparin complexes in the K/B×N mouse arthritis model and connect our mouse findings with RA pathophysiology.
PMCID: PMC2610352  PMID: 19109198
mast cell; rheumatoid arthritis; inflammation; chemokines; transgenic/knockout mice
21.  Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN) 
Clinical immunology (Orlando, Fla.)  2008;129(1):115-122.
Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model.
Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−,GRK2+/−, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays.
Both GRK6−/− and GRK2+/− mice had increased arthritis disease severity (p<0.001); whereas GRK5−/− was not different from controls. Acute weight loss was enhanced in GRK6−/− and GRK2+/− mice (p<0.001, days 3–10). However, GRK6−/− mice uniquely had more weight loss (>10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro.
GRK6 and - 2, but not GRK5,are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.
PMCID: PMC2637521  PMID: 18662895
22.  Human neutrophil FcγRIIA regulation by C5aR promotes inflammatory arthritis in mice 
Arthritis and Rheumatism  2011;63(2):467-478.
Rheumatoid arthritis culminates in joint destruction that in mouse models of disease, is supported by innate immune molecules including FcγRs and complement. However, the results may not predict outcomes in humans given the structural differences between murine and human activating FcγRs on neutrophils, a prominent component of joint exudates. In this study, we examined the role of the human neutrophil FcγRIIA in the development of arthritis and probed the underlying mechanism by which FcγRIIA initiated disease.
K/BxN serum transfer-induced arthritis was examined in mice that express FcγRIIA on neutrophils but lack their own activating FcγRs (γ-chain-deficient). The role of mast cells, complement (C3 and C5a) and CD18 integrins in FcγRIIA-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies respectively. Cross-talk between C5aR and FcγRIIA on neutrophils was evaluated in vitro.
Neutrophil FcγRIIA expression was sufficient to restore susceptibility to K/BxN serum induced neutrophil recruitment, synovitis and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on CD18 integrin LFA-1 and C5aR. C5aR in addition significantly enhanced FcγRIIA mediated phagocytosis and oxidative burst in vitro.
Human and murine activating FcγRs on neutrophils are not functionally equivalent, and in humans may play a primary role in arthritis. Cross-talk between neutrophil C5aR and FcγRIIA is essential for disease thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.
PMCID: PMC3128197  PMID: 21280001
23.  Hypogalactosylation of serum N-glycans fails to predict clinical response to methotrexate and TNF inhibition in rheumatoid arthritis 
Rheumatoid arthritis (RA) is associated with hypogalactosylation of immunoglobulin G (IgG). We examined whether a proxy measure for galactosylation of IgG N-glycans could predict response to therapy or was differentially affected by methotrexate (MTX) or TNF blockade.
Using a previously defined normal phase high-performance liquid chromatography approach, we ascertained the galactosylation status of whole serum N-glycans in two well-defined RA clinical cohorts: the Autoimmune Biomarkers Collaborative Network (n = 98) and Nested I (n = 64). The ratio of agalactosylated to monogalactosylated N-glycans in serum (sG0/G1) was determined before and during therapy with MTX or TNF inhibition and correlated with anticitrullinated peptide antibody (ACPA) status and clinical response as assessed by 28-joint Disease Activity Score utilizing C-reactive peptide and European League Against Rheumatism response criteria.
RA patients from both cohorts exhibited elevation of sG0/G1 at baseline. Improvement in clinical scores correlated with a reduction in sG0/G1 (Spearman's ρ = 0.31 to 0.37; P < 0.05 for each cohort). However, pretreatment sG0/G1 was not predictive of clinical response. Changes in sG0/G1 were similar in the MTX and TNF inhibitor groups. Corrected for disease activity, ACPA positivity correlated with higher sG0/G1.
Baseline serum N-glycan hypogalactosylation, an index previously correlated with hypogalactosylation of IgG N-glycans, did not distinguish patients with rheumatoid arthritis who were likely to experience a favorable clinical response to MTX or TNF blockade. Clinical improvement was associated with partial glycan normalization. ACPA-positive patients demonstrated enhanced N-glycan aberrancy compared with ACPA-negative patients.
PMCID: PMC3446410  PMID: 22390545
24.  Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship with symptoms 
Arthritis and Rheumatism  2011;63(2):391-400.
Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.
Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
PMCID: PMC3260472  PMID: 21279996
Meniscectomy; meniscal injury; inflammation; synovium; synovitis
25.  The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation 
Pereyra, Florencia | Jia, Xiaoming | McLaren, Paul J. | Telenti, Amalio | de Bakker, Paul I.W. | Walker, Bruce D. | Jia, Xiaoming | McLaren, Paul J. | Ripke, Stephan | Brumme, Chanson J. | Pulit, Sara L. | Telenti, Amalio | Carrington, Mary | Kadie, Carl M. | Carlson, Jonathan M. | Heckerman, David | de Bakker, Paul I.W. | Pereyra, Florencia | de Bakker, Paul I.W. | Graham, Robert R. | Plenge, Robert M. | Deeks, Steven G. | Walker, Bruce D. | Gianniny, Lauren | Crawford, Gabriel | Sullivan, Jordan | Gonzalez, Elena | Davies, Leela | Camargo, Amy | Moore, Jamie M. | Beattie, Nicole | Gupta, Supriya | Crenshaw, Andrew | Burtt, Noël P. | Guiducci, Candace | Gupta, Namrata | Carrington, Mary | Gao, Xiaojiang | Qi, Ying | Yuki, Yuko | Pereyra, Florencia | Piechocka-Trocha, Alicja | Cutrell, Emily | Rosenberg, Rachel | Moss, Kristin L. | Lemay, Paul | O’Leary, Jessica | Schaefer, Todd | Verma, Pranshu | Toth, Ildiko | Block, Brian | Baker, Brett | Rothchild, Alissa | Lian, Jeffrey | Proudfoot, Jacqueline | Alvino, Donna Marie L. | Vine, Seanna | Addo, Marylyn M. | Allen, Todd M. | Altfeld, Marcus | Henn, Matthew R. | Le Gall, Sylvie | Streeck, Hendrik | Walker, Bruce D. | Haas, David W. | Kuritzkes, Daniel R. | Robbins, Gregory K. | Shafer, Robert W. | Gulick, Roy M. | Shikuma, Cecilia M. | Haubrich, Richard | Riddler, Sharon | Sax, Paul E. | Daar, Eric S. | Ribaudo, Heather J. | Agan, Brian | Agarwal, Shanu | Ahern, Richard L. | Allen, Brady L. | Altidor, Sherly | Altschuler, Eric L. | Ambardar, Sujata | Anastos, Kathryn | Anderson, Ben | Anderson, Val | Andrady, Ushan | Antoniskis, Diana | Bangsberg, David | Barbaro, Daniel | Barrie, William | Bartczak, J. | Barton, Simon | Basden, Patricia | Basgoz, Nesli | Bazner, Suzane | Bellos, Nicholaos C. | Benson, Anne M. | Berger, Judith | Bernard, Nicole F. | Bernard, Annette M. | Birch, Christopher | Bodner, Stanley J. | Bolan, Robert K. | Boudreaux, Emilie T. | Bradley, Meg | Braun, James F. | Brndjar, Jon E. | Brown, Stephen J. | Brown, Katherine | Brown, Sheldon T. | Burack, Jedidiah | Bush, Larry M. | Cafaro, Virginia | Campbell, Omobolaji | Campbell, John | Carlson, Robert H. | Carmichael, J. Kevin | Casey, Kathleen K. | Cavacuiti, Chris | Celestin, Gregory | Chambers, Steven T. | Chez, Nancy | Chirch, Lisa M. | Cimoch, Paul J. | Cohen, Daniel | Cohn, Lillian E. | Conway, Brian | Cooper, David A. | Cornelson, Brian | Cox, David T. | Cristofano, Michael V. | Cuchural, George | Czartoski, Julie L. | Dahman, Joseph M. | Daly, Jennifer S. | Davis, Benjamin T. | Davis, Kristine | Davod, Sheila M. | Deeks, Steven G. | DeJesus, Edwin | Dietz, Craig A. | Dunham, Eleanor | Dunn, Michael E. | Ellerin, Todd B. | Eron, Joseph J. | Fangman, John J.W. | Farel, Claire E. | Ferlazzo, Helen | Fidler, Sarah | Fleenor-Ford, Anita | Frankel, Renee | Freedberg, Kenneth A. | French, Neel K. | Fuchs, Jonathan D. | Fuller, Jon D. | Gaberman, Jonna | Gallant, Joel E. | Gandhi, Rajesh T. | Garcia, Efrain | Garmon, Donald | Gathe, Joseph C. | Gaultier, Cyril R. | Gebre, Wondwoosen | Gilman, Frank D. | Gilson, Ian | Goepfert, Paul A. | Gottlieb, Michael S. | Goulston, Claudia | Groger, Richard K. | Gurley, T. Douglas | Haber, Stuart | Hardwicke, Robin | Hardy, W. David | Harrigan, P. Richard | Hawkins, Trevor N. | Heath, Sonya | Hecht, Frederick M. | Henry, W. Keith | Hladek, Melissa | Hoffman, Robert P. | Horton, James M. | Hsu, Ricky K. | Huhn, Gregory D. | Hunt, Peter | Hupert, Mark J. | Illeman, Mark L. | Jaeger, Hans | Jellinger, Robert M. | John, Mina | Johnson, Jennifer A. | Johnson, Kristin L. | Johnson, Heather | Johnson, Kay | Joly, Jennifer | Jordan, Wilbert C. | Kauffman, Carol A. | Khanlou, Homayoon | Killian, Robert K. | Kim, Arthur Y. | Kim, David D. | Kinder, Clifford A. | Kirchner, Jeffrey T. | Kogelman, Laura | Kojic, Erna Milunka | Korthuis, P. Todd | Kurisu, Wayne | Kwon, Douglas S. | LaMar, Melissa | Lampiris, Harry | Lanzafame, Massimiliano | Lederman, Michael M. | Lee, David M. | Lee, Jean M.L. | Lee, Marah J. | Lee, Edward T.Y. | Lemoine, Janice | Levy, Jay A. | Llibre, Josep M. | Liguori, Michael A. | Little, Susan J. | Liu, Anne Y. | Lopez, Alvaro J. | Loutfy, Mono R. | Loy, Dawn | Mohammed, Debbie Y. | Man, Alan | Mansour, Michael K. | Marconi, Vincent C. | Markowitz, Martin | Marques, Rui | Martin, Jeffrey N. | Martin, Harold L. | Mayer, Kenneth Hugh | McElrath, M. Juliana | McGhee, Theresa A. | McGovern, Barbara H. | McGowan, Katherine | McIntyre, Dawn | Mcleod, Gavin X. | Menezes, Prema | Mesa, Greg | Metroka, Craig E. | Meyer-Olson, Dirk | Miller, Andy O. | Montgomery, Kate | Mounzer, Karam C. | Nagami, Ellen H. | Nagin, Iris | Nahass, Ronald G. | Nelson, Margret O. | Nielsen, Craig | Norene, David L. | O’Connor, David H. | Ojikutu, Bisola O. | Okulicz, Jason | Oladehin, Olakunle O. | Oldfield, Edward C. | Olender, Susan A. | Ostrowski, Mario | Owen, William F. | Pae, Eunice | Parsonnet, Jeffrey | Pavlatos, Andrew M. | Perlmutter, Aaron M. | Pierce, Michael N. | Pincus, Jonathan M. | Pisani, Leandro | Price, Lawrence Jay | Proia, Laurie | Prokesch, Richard C. | Pujet, Heather Calderon | Ramgopal, Moti | Rathod, Almas | Rausch, Michael | Ravishankar, J. | Rhame, Frank S. | Richards, Constance Shamuyarira | Richman, Douglas D. | Robbins, Gregory K. | Rodes, Berta | Rodriguez, Milagros | Rose, Richard C. | Rosenberg, Eric S. | Rosenthal, Daniel | Ross, Polly E. | Rubin, David S. | Rumbaugh, Elease | Saenz, Luis | Salvaggio, Michelle R. | Sanchez, William C. | Sanjana, Veeraf M. | Santiago, Steven | Schmidt, Wolfgang | Schuitemaker, Hanneke | Sestak, Philip M. | Shalit, Peter | Shay, William | Shirvani, Vivian N. | Silebi, Vanessa I. | Sizemore, James M. | Skolnik, Paul R. | Sokol-Anderson, Marcia | Sosman, James M. | Stabile, Paul | Stapleton, Jack T. | Starrett, Sheree | Stein, Francine | Stellbrink, Hans-Jurgen | Sterman, F. Lisa | Stone, Valerie E. | Stone, David R. | Tambussi, Giuseppe | Taplitz, Randy A. | Tedaldi, Ellen M. | Telenti, Amalio | Theisen, William | Torres, Richard | Tosiello, Lorraine | Tremblay, Cecile | Tribble, Marc A. | Trinh, Phuong D. | Tsao, Alice | Ueda, Peggy | Vaccaro, Anthony | Valadas, Emilia | Vanig, Thanes J. | Vecino, Isabel | Vega, Vilma M. | Veikley, Wenoah | Wade, Barbara H. | Walworth, Charles | Wanidworanun, Chingchai | Ward, Douglas J. | Warner, Daniel A. | Weber, Robert D. | Webster, Duncan | Weis, Steve | Wheeler, David A. | White, David J. | Wilkins, Ed | Winston, Alan | Wlodaver, Clifford G. | Wout, Angelique van’t | Wright, David P. | Yang, Otto O. | Yurdin, David L. | Zabukovic, Brandon W. | Zachary, Kimon C. | Zeeman, Beth | Zhao, Meng
Science (New York, N.Y.)  2010;330(6010):1551-1557.
Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection.
PMCID: PMC3235490  PMID: 21051598

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