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1.  Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation 
Introduction
We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined.
Methods
Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed.
Results
Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation.
Conclusions
These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA.
doi:10.1186/ar4456
PMCID: PMC3978694  PMID: 24467809
2.  Gp96 Perpetuates the Persistent Inflammation of Rheumatoid Arthritis 
Arthritis and rheumatism  2012;64(11):3638-3648.
Objective
The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. This study was performed to determine the contribution of endogenous gp96 in TLR-mediated macrophage activation in RA.
Methods
RA synovial fluids (SFs) were employed to activate macrophages and HEK-TLR2 and HEK-TLR4 cells. Neutralizing antibodies to TLR2, TLR4 and gp96 were employed to inhibit activation. RA SF macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of TNFα or IL-8 mRNA. Arthritis was induced employing the K/BxN serum transfer model, gp96 expression determined by Immunoblot analysis, ELISA and immunohistochemistry. The arthritis was treated with neutralizing anit-gp96 or control serum.
Results
RA SF induced the activation of macrophages and HEK-TLR2 and HEK-TLR4 cells. RA SF-induced macrophage and HEK-TLR2 activation was suppressed by neutralizing anti-gp96 antibody only when high (>800 ng/ml), but not low (<400 ng/ml), concentrations of gp96 were present. Neutralization of RA SF macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting its role in RA, joint tissue gp96 expression was induced in the K/BxN serum transfer model of RA, and neutralizing antibodies to gp96, ameliorated joint inflammation on clinical and histologic examination.
Conclusions
These observations support the role of gp96 as an endogenous TLR2 ligand in RA and identify the TLR2 pathway as a therapeutic target.
doi:10.1002/art.34610
PMCID: PMC3473144  PMID: 22777994
3.  Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES 
Arthritis and rheumatism  2005;52(6):1907-1919.
Objective
CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1α (MIP-1α; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat.
Methods
Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA.
Results
Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1α and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor α, interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint.
Conclusion
The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
doi:10.1002/art.21033
PMCID: PMC1282452  PMID: 15934086
4.  Myeloid Cell-Specific Fas Expression is Required to Prevent Systemic Autoimmunity 
Arthritis and Rheumatism  2012;64(3):808-820.
OBJECTIVE
The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway. While the role that Fas plays in mediating lymphoproliferation has been extensively investigated, the impact of myeloid cell-specific loss of Fas has yet to be examined.
METHODS
Mice with Fas flanked by loxP sites (Fasflox/flox) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (CreLysM), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for CreLysMFasflox/flox mice was verified by real-time PCR and flow cytometric analysis. Flow cytometric analysis was also employed to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood and spleen. Luminex-based assays and ELISAs were used to measure serum cytokine/chemokine and immunglobulin levels. Immunohistochemical and immunofluorescent analyses were utilized to examine renal damage or dysfunction.
RESULTS
CreLysMFasflox/flox mice exhibited an SLE-like disease including leukocytosis, splenomegaly, hypergammaglobulinemia, anti-nuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1low and Gr-1intermediate blood monocytes and splenic macrophages, and in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in CreLysMFasflox/flox mice.
CONCLUSION
Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor systemic autoimmunity.
doi:10.1002/art.34317
PMCID: PMC3290732  PMID: 22143975
5.  The Cyclin Dependent Kinase Inhibitor p21 Is Essential for Resolution of Murine Inflammatory Arthritis via its C-Terminal Domain 
Arthritis and Rheumatism  2012;64(1):141-152.
Objective
The mechanism responsible for persistent inflammation of the synovium that occurs in patients with rheumatoid arthritis (RA) is unknown. Previously, we were the first to demonstrate that expression of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. Here, we sought to determine the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis.
Methods
Experimental arthritis was induced in WT or p21−/− (C57BL/6) mice using the K/BxN serum transfer induced model. p21-peptide mimetics were administered to mice as a prophylactic for arthritis development. LPS-induced cytokine and signal transduction pathways were examined in macrophages that were treated with p21-peptide mimetics using Luminex-based assays, flow cytometry, or ELISAs.
Results
p21−/− mice exhibit enhanced and sustained development of experimental inflammatory arthritis, which is associated with markedly increased numbers of macrophages and severe articular destruction. Administration of a p21-peptide mimetic suppresses activation of macrophages and reduces the severity of experimental arthritis only in p21-intact mice. Mechanistically, treatment with the p21-peptide mimetic leads to activation of the serine/threonine kinase Akt and subsequent reduction in the activated isoform of mitogen-activated protein kinase p38 in macrophages.
Conclusion
These data are the first to reveal that p21 plays an important role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.
doi:10.1002/art.33311
PMCID: PMC3253189  PMID: 21898359
6.  Development of a new humanized mouse model to study acute inflammatory arthritis 
Background
Substantial advances have been generated in understanding the pathogenesis of rheumatoid arthritis (RA). Current murine models of RA-like disease have provided great insights into the molecular mechanism of inflammatory arthritis due to the use of genetically deficient or transgenic mice. However, these studies are limited by differences that exist between human and murine immune systems. Thus, the development of an animal model that utilizes human immune cells, will afford the opportunity to study their function in the initiation and propagation of inflammatory arthritis.
Methods
One to two-day old irradiated NOD-scid IL2rγnull (NSG) mice were reconstituted with human CD34+ cord blood stem cells. Leukocytes were analyzed by flow cytometry and circulating antibodies were determined by ELISA. Arthritis was induced by injecting complete Freund’s adjuvant into knee or ankle joints. Mice were also treated with the TNF inhibitor, Etanercept, or PBS and joints were analyzed histologically.
Results
Humanized mice were established with high reconstitution rates and were able to spontaneously produce human immunoglobulins as well as specific IgG in response to immunization. Intraperitoneal injection of thioglycolate or injection of complete Freund’s adjuvant into joints resulted in migration of human immune cells to the injected sites. Arthritic humanized mice treated with Etanercept had markedly less inflammation, which was associated with decreased total numbers of human CD45+ cells, including human lymphocytes and neutrophils.
Conclusions
The humanized mouse model is a new model to study inflammatory arthritis disease using human leukocytes without rejection of engrafted tissue. Future studies may adapt this system to incorporate RA patient cord blood and develop a chimeric animal model of inflammatory arthritis using genetically predisposed immune cells.
doi:10.1186/1479-5876-10-190
PMCID: PMC3480927  PMID: 22974474
Humanized mouse; Leukocytes; Rheumatoid arthritis; Etanercept
7.  Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis 
Introduction
Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.
Methods
Immunohistology and enzyme-linked immunosorbent assays (ELISAs) were used to determine chemokine and chemokine receptor expression in the skin and serum, respectively, of SSc and normal patients. Endothelial cells (ECs) were isolated from SSc skin biopsies and chemokine and chemokine receptor expression was determined by quantitative PCR and immunofluorescence staining.
Results
Antiangiogenic IP-10/CXCL10 and MIG/CXCL9 were elevated in SSc serum and highly expressed in SSc skin. However, CXCR3, the receptor for these chemokines, was decreased on ECs in SSc vs. normal skin. CXCL16 was elevated in SSc serum and increased in SSc patients with early disease, pulmonary arterial hypertension, and those that died during the 36 months of the study. In addition, its receptor CXCR6 was overexpressed on ECs in SSc skin. At the mRNA and protein levels, CXCR3 was decreased while CXCR6 was increased on SSc ECs vs. human microvascular endothelial cells (HMVECs).
Conclusions
These results show that while the expression of MIG/CXCL9 and IP-10/CXCL10 are elevated in SSc serum, the expression of CXCR3 is downregulated on SSc dermal ECs. In contrast, CXCL16 and CXCR6 are elevated in SSc serum and on SSc dermal ECs, respectively. In all, these findings suggest angiogenic chemokine receptor expression is likely regulated in an effort to promote angiogenesis in SSc skin.
doi:10.1186/ar3242
PMCID: PMC3241362  PMID: 21303517
8.  Synovial Inflammation in Patients with Osteonecrosis of the Femoral Head 
Much of the work aimed at elucidating the pathogenesis of osteonecrosis (ON) of the femoral head has focused on bone blood supply, with little attention to the surrounding synovial tissue (ST). We hypothesized that patients with ON exhibit synovial inflammation. Using immunohistological techniques, we found that a large population of patients with ON had synovial inflammation. Moreover, a population of ON patients had inflamed ST without having an inflammatory disease co-morbidity. The inflammatory infiltrate in these patients comprised primarily CD4+ T cells and CD68+ macrophages, the latter of which expressed increased levels of cellular adhesion molecules. Our results suggest the presence of a previously unrecognized population of ON patients without a diagnosed inflammatory co-morbidity and a highly inflammed synovium consisting primarily of a macrophage and CD4+ T-cell infiltrate.
doi:10.1111/j.1752-8062.2009.00133.x
PMCID: PMC2925228  PMID: 20443906
osteonecrosis; synovium; inflammation; macrophage; adhesion molecules
9.  Distinct Human Papillomavirus Type 16 Methylomes in Cervical Cells at Different Stages of Premalignancy 
Virology  2009;389(1-2):100-107.
Human papillomavirus (HPV) gene expression is dramatically altered during cervical carcinogenesis. Because dysregulated genes frequently show abnormal patterns of DNA methylation, we hypothesized that comprehensive mapping of the HPV methylomes in cervical samples at different stages of progression would reveal patterns of clinical significance. To test this hypothesis, thirteen HPV16-positive samples were obtained from women undergoing routine cervical cancer screening. Complete methylation data were obtained for 98.7% of the HPV16 CpGs in all samples by bisulfite-sequencing. Most HPV16 CpGs were unmethylated or methylated in only one sample. The other CpGs were methylated at levels ranging from 11% to 100% of the HPV16 copies per sample. The results showed three major patterns and two variants of one pattern. The patterns showed minimal or no methylation (A), low level methylation in the E1 and E6 genes (B), and high level methylation at many CpGs in the E5/L2/L1 region (C). Generally, pattern A was associated with negative cytology, pattern B with low-grade lesions, and pattern C with high-grade lesions. The severity of the cervical lesions was then ranked by the HPV16 DNA methylation patterns and, independently, by the pathologic diagnoses. Statistical analysis of the two rating methods showed highly significant agreement. In conclusion, analysis of the HPV16 DNA methylomes in clinical samples of cervical cells led to the identification of distinct methylation patterns which, after validation in larger studies, could have potential utility as biomarkers of neoplastic cervical progression.
doi:10.1016/j.virol.2009.03.029
PMCID: PMC2918277  PMID: 19443004
10.  Junctional Adhesion Molecule C Mediates Leukocyte Adhesion to Rheumatoid Arthritis Synovium 
Arthritis and rheumatism  2008;58(10):3020-3029.
Objective
Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint.
Methods
Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed.
Results
JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C.
Conclusion
Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA.
doi:10.1002/art.23867
PMCID: PMC2911024  PMID: 18821692
11.  Junctional adhesion molecule-A is abnormally expressed in diffuse cutaneous systemic sclerosis skin and mediates myeloid cell adhesion 
Annals of the rheumatic diseases  2010;69(1):249-254.
Objective
To investigate the role of Junctional adhesion molecule A (JAM-A) in the pathogenesis of systemic sclerosis (SSc).
Methods
Biopsies from proximal and distal arm skin and serum were obtained from patients with SSc and normal (NL) volunteers. To determine the expression of JAM-A on SSc dermal fibroblasts and in SSc skin, cell surface ELISAs and immunohistology were performed. An ELISA was designed to determine the amount of soluble JAM-A (sJAM-A) in serum. Myeloid U937 cell-SSc dermal fibroblast and skin adhesion assays were performed to determine the role of JAM-A in myeloid cell adhesion.
Results
The stratum granulosum and dermal endothelial cells (ECs) from distal arm SSc skin exhibited significantly decreased expression of JAM-A compared to NL. However, sJAM-A was elevated in the serum of patients with SSc compared to NL. Conversely, JAM-A was increased on the surface of SSc compared to NL dermal fibroblasts. JAM-A accounted for a significant portion of U937 binding to SSc dermal fibroblasts. In addition, JAM-A contributed to U937 adhesion to both distal and proximal SSc skin.
Conclusions
JAM-A expression is dysregulated in SSc skin. Decreased expression of JAM-A on SSc ECs may result in a reduced response to proangiogenic basic fibroblast growth factor. While increased JAM-A expression on SSc fibroblasts may serve to retain myeloid cells, which in turn secrete angiogenic factors.
doi:10.1136/ard.2008.102624
PMCID: PMC2795028  PMID: 19153103
Systemic sclerosis; Scleroderma; JAM-A; Cell adhesion
12.  Bim-BH3 mimetic therapy is effective at suppressing inflammatory arthritis through the activation of myeloid cell apoptosis 
Arthritis and rheumatism  2010;62(2):441-451.
Objective
Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies which activate the apoptotic cascade may have potential as a future therapy for RA, however few therapeutics fit this category. Recently, therapies that mimic the action of Bcl-2 homology 3 (BH3) domain-only proteins such as Bim have shown success in preclinical studies of cancer but their potential in autoimmune disease is unknown.
Methods
Synovial tissue from RA and osteoarthritis (OA) patients were analyzed for expression of Bim and CD68 using immunohistochemistry. Macrophages from mice lacking (Bim−/−) were examined for response to lipopolysaccharide (LPS) using flow cytometry, real time PCR, ELISA, and immunoblot analysis. Bim−/− mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic and as a therapeutic. Edema of the ankles and histopathogical analysis of ankle sections were used to determine severity of arthritis, cellular composition, and apoptosis.
Results
The expression of Bim was reduced in RA synovial tissue as compared to controls, particularly in macrophages. Bim−/− macrophages displayed elevated expression of markers of inflammation and secreted more IL-1β following stimulation with LPS or thioglycollate. TAT-BH3 ameliorated arthritis development, reduced the number of myeloid cells in the joint, and enhanced apoptosis without inducing cytotoxicity.
Conclusion
These data demonstrate that BH3 mimetic therapy may have significant potential for RA treatment.
doi:10.1002/art.27198
PMCID: PMC2848986  PMID: 20112357
Bim; arthritis; macrophages; apoptosis
13.  Nitric Oxide: Perspectives and Emerging Studies of a Well Known Cytotoxin 
The free radical nitric oxide (NO•) is known to play a dual role in human physiology and pathophysiology. At low levels, NO• can protect cells; however, at higher levels, NO• is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO• in cancer has to date been tissue-specific, we herein review underlying commonalities of NO• which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO• in human physiology and pathophysiology, including the very important relationship between NO• and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO• in three main areas of epigenetics—DNA methylation, microRNAs, and histone modifications—is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO• (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO• in cancer.
doi:10.3390/ijms11072715
PMCID: PMC2920563  PMID: 20717533
nitric oxide; epigenetics; cytotoxicity; high NO adaptation; oncogenetic
14.  Hemolysin and the Multifunctional Autoprocessing RTX Toxin Are Virulence Factors during Intestinal Infection of Mice with Vibrio cholerae El Tor O1 Strains▿  
Infection and Immunity  2007;75(10):5035-5042.
The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.
doi:10.1128/IAI.00506-07
PMCID: PMC2044521  PMID: 17698573
15.  CXCL16-Mediated Cell Recruitment to Rheumatoid Arthritis Synovial Tissue and Murine Lymph Nodes Is Dependent Upon the MAPK Pathway 
Arthritis and rheumatism  2006;54(3):765-778.
Objective
Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor α (TNFα), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process.
Methods
Using a human RA ST–SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo.
Results
CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFα. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFα in vitro, suggesting that CXCL16 protein may function in large part independently of TNFα.
Conclusion
Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.
doi:10.1002/art.21662
PMCID: PMC1472704  PMID: 16508941
16.  Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues 
The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.
doi:10.1186/ar2318
PMCID: PMC2212559  PMID: 17963503
17.  Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis 
Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1β in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.
doi:10.1186/ar2204
PMCID: PMC2206343  PMID: 17509138
18.  Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
doi:10.1186/ar2021
PMCID: PMC1779381  PMID: 16872482
19.  Role of Toll-Like Receptor 4 in the Proinflammatory Response to Vibrio cholerae O1 El Tor Strains Deficient in Production of Cholera Toxin and Accessory Toxins  
Infection and Immunity  2005;73(9):6157-6164.
Following intranasal inoculation, Vibrio cholerae KFV101 (ΔctxAB ΔhapA ΔhlyA ΔrtxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1β (IL-1β) in mice, similar to what occurs with isogenic strain P4 (ΔctxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.
doi:10.1128/IAI.73.9.6157-6164.2005
PMCID: PMC1231125  PMID: 16113340
20.  Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line 
Background
Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NOX) have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs) and apoptosis in cultured tumor cell lines.
Methods
Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX via donor compounds. Production of NOX was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay), and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay.
Results
Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds.
Conclusions
Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when developing NOX-based chemotherapeutic agents.
doi:10.1186/1477-3163-3-16
PMCID: PMC544845  PMID: 15617570
Nitric Oxide; DNA Strand Breaks
21.  The Contribution of Accessory Toxins of Vibrio cholerae O1 El Tor to the Proinflammatory Response in a Murine Pulmonary Cholera Model 
The Journal of Experimental Medicine  2002;195(11):1455-1462.
The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.
doi:10.1084/jem.20020318
PMCID: PMC2193536  PMID: 12045243
Vibrio cholerae; inflammation; RTX toxin; hemolysin; hemagglutinin/protease
22.  Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue 
Arthritis Research  2002;4(3):201-208.
Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA).
Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis.
In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.
PMCID: PMC111023  PMID: 12010571
angiopoietin; mRNA; rheumatoid arthritis; Tie receptor expression

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