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1.
BMJ ; 362: k3103, 2018 07 13.
Article in English | MEDLINE | ID: mdl-30006477
2.
Eur J Immunol ; 31(8): 2421-30, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11500826

ABSTRACT

Peripheral blood-derived monocytes spontaneously undergo apoptosis mediated by Fas-Fas ligand (FasL) interactions. Activation of monocytes by LPS or TNF-alpha prevents spontaneous monocyte apoptosis through an unknown mechanism. Here, we demonstrate that LPS and TNF-alpha up-regulate Flip and suppress spontaneous Fas-FasL mediated monocyte apoptosis and caspase 8 and 3 activation. Flip was responsible for this protection, since inhibition of Flip by antisense oligonucleotides in the presence of LPS or TNF-alpha restored monocyte sensitivity to spontaneous apoptosis. We also investigated whether the PI3K pathway contributes to the suppression of spontaneous monocyte apoptosis mediated by LPS and TNF-alpha. Monocytes treated with a reversible PI3K inhibitor (LY294002) displayed enhanced apoptosis, while LPS and TNF-alpha partially protected against apoptosis mediated by LY294002. However, direct suppression of Fas-FasL interactions by addition of neutralizing anti-FasL antibody did not further suppress LY294002-induced apoptosis in the presence of LPS or TNF-alpha. Collectively, these data demonstrate that LPS or TNF-alpha protect monocytes from death receptor-mediated apoptosis through the up-regulation of Flip, but not apoptosis initiated by inhibition of the PI3K pathway.


Subject(s)
Apoptosis , Homeostasis , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins/metabolism , Monocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , fas Receptor/metabolism , Animals , Antibodies/pharmacology , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Chromones/antagonists & inhibitors , Chromones/pharmacology , Enzyme Activation/drug effects , Fas Ligand Protein , Homeostasis/drug effects , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/antagonists & inhibitors , Mitochondria/drug effects , Mitochondria/metabolism , Monocytes/cytology , Monocytes/drug effects , Monocytes/enzymology , Morpholines/antagonists & inhibitors , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects
3.
J Exp Med ; 194(2): 113-26, 2001 Jul 16.
Article in English | MEDLINE | ID: mdl-11457886

ABSTRACT

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain-containing 5' inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-x(L). Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-kappaB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-kappaB, or Bad.


Subject(s)
Macrophages/metabolism , Monocytes/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins , Animals , Apoptosis/drug effects , Base Sequence , Carrier Proteins/metabolism , Caspase Inhibitors , Caspases/metabolism , Cell Differentiation , Cell Survival , Chromones/pharmacology , DNA Primers/genetics , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Macrophages/cytology , Macrophages/drug effects , Mice , Mitochondria/metabolism , Monocytes/cytology , Monocytes/drug effects , Morpholines/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt , bcl-Associated Death Protein
4.
Glycobiology ; 11(6): 505-13, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11445555

ABSTRACT

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides by using nano-electrospray ionization mass spectrometry (nESI-MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1--3 min. Hence, our data indicated that as little as 3 x 10(-15) mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[(35)S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [(35)S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.


Subject(s)
Heparitin Sulfate/chemistry , Oligosaccharides/chemistry , Spectrometry, Mass, Electrospray Ionization , Carbohydrate Sequence , Molecular Sequence Data
5.
Arthritis Rheum ; 44(5): 1022-32, 2001 May.
Article in English | MEDLINE | ID: mdl-11352233

ABSTRACT

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.


Subject(s)
Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Receptors, Chemokine/biosynthesis , Synovial Fluid/metabolism , Synovial Membrane/metabolism , Adult , Chemokine CCL2/immunology , Chemokine CCL2/metabolism , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Female , Flow Cytometry , Humans , Macrophages/immunology , Macrophages/metabolism , Male , Monocytes/immunology , Monocytes/metabolism , Receptors, CCR6 , Receptors, CXCR3 , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/immunology , Receptors, CXCR5 , Receptors, Chemokine/immunology , Receptors, Cytokine/biosynthesis , Receptors, Cytokine/immunology , Receptors, Interleukin-8A/biosynthesis , Receptors, Interleukin-8A/immunology , Receptors, Interleukin-8B/biosynthesis , Receptors, Interleukin-8B/immunology , Synovial Fluid/cytology , Synovial Fluid/immunology , Synovial Membrane/cytology , Synovial Membrane/immunology
6.
Gene Ther ; 8(3): 223-31, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11313794

ABSTRACT

Macrophage-derived TNF alpha is a critical mediator of inflammation and destruction in diseases such as rheumatoid arthritis and Crohn's disease. These studies were undertaken to develop an effective adenovirus-based strategy to specifically suppress TNF alpha in primary human macrophages. A variety of promoters and LTRs were evaluated for effective expression in the macrophage cell line RAW 264.7. The CMV promoter and the Visna LTR were the most strongly expressed and were therefore used to drive the expression of TNF alpha antisense fragments. In transient transfection assays, the antisense fragment terminating at the 3' end of the first exon (216 bp) was superior to the others (70 and 750 bp), when expressed under the control of either the CMV promoter or the Visna LTR. Adenoviral vectors expressing the 216 bp TNF alpha antisense fragment, controlled by the CMV promoter or the Visna LTR, were both effective at suppressing LPS-induced TNF alpha secretion by primary human macrophages. However, the Visna LTR was more effective not only at suppressing LPS-induced TNF alpha secretion, but also IL-6, which is highly sensitive to TNF alpha secretion. These results demonstrate that effective, specific, suppression of TNF alpha in macrophages is possible, employing a directed antisense approach and a promoter system that is highly efficient in human macrophages.


Subject(s)
Antisense Elements (Genetics)/genetics , Macrophages/immunology , Promoter Regions, Genetic/genetics , Tumor Necrosis Factor-alpha/genetics , Adenoviridae/genetics , Animals , Cell Line , Cytomegalovirus/genetics , Humans , Interleukin-6/metabolism , Lipopolysaccharides/immunology , Mice , Plasmids , Transfection , Tumor Necrosis Factor-alpha/metabolism
7.
Arthritis Rheum ; 44(1): 21-30, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11212162

ABSTRACT

OBJECTIVE: The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHODS: The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. RESULTS: CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. CONCLUSION: These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.


Subject(s)
Adaptor Proteins, Signal Transducing , Arthritis, Rheumatoid/pathology , Carrier Proteins/analysis , Macrophages/chemistry , Serpins/biosynthesis , Viral Proteins , Apoptosis/drug effects , Caspase 8 , Caspase 9 , Caspases/biosynthesis , Cysteine Proteinase Inhibitors/biosynthesis , Fas-Associated Death Domain Protein , Fluorescent Antibody Technique , Monocytes/cytology , Synovial Membrane/cytology , fas Receptor/analysis
8.
Arthritis Rheum ; 44(12): 2750-60, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11762935

ABSTRACT

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.


Subject(s)
Arthritis, Rheumatoid/immunology , Joints/immunology , Receptors, Chemokine/immunology , Synovial Fluid/immunology , CD3 Complex/analysis , CD4-Positive T-Lymphocytes/chemistry , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/chemistry , CD8-Positive T-Lymphocytes/immunology , CX3C Chemokine Receptor 1 , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunologic Memory/immunology , Joints/chemistry , Receptors, CCR4 , Receptors, CCR5/analysis , Receptors, CCR5/immunology , Receptors, CXCR3 , Receptors, Chemokine/analysis , Receptors, Cytokine/analysis , Receptors, Cytokine/immunology , Receptors, HIV/analysis , Receptors, HIV/immunology , Synovial Fluid/chemistry
9.
Arthritis Rheum ; 44(12): 2899-908, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11762951

ABSTRACT

OBJECTIVE: To examine the relationship between apoptosis and the expression of antiapoptotic proteins in the pathogenesis of experimental inflammatory arthritis. METHODS: Clinical and histologic assessment of adjuvant-induced arthritis (AIA) was performed over a 42-day period. The induction of apoptosis was measured by TUNEL analysis, and the antiapoptotic proteins, Bcl-2 and FLIP, were examined by immunohistochemistry with the use of monospecific antibodies. The percentage of Bcl-2- and FLIP-positive cells was correlated with histologic markers of AIA. RESULTS: Arthritis developed by day 14 following adjuvant injection. Few TUNEL-positive cells were observed between days 0 and 21, indicating that apoptosis did not occur at these time points. An increase in the number of TUNEL-positive cells was observed at day 28, particularly outside sites of cartilage or bone erosion, which dramatically declined by day 35. Immunohistochemical analyses of Bcl-2 and FLIP revealed that the synovium was positive for Bcl-2 and FLIP on day 0. On day 14, Bcl-2 was present at the sites of early erosions and correlated with the erosion and inflammation scores. FLIP was also highly expressed at sites of erosion and was localized to the pannus starting on day 21. Although TUNEL positivity peaked at day 28, a time point in which Bcl-2 and FLIP were present, the areas that displayed intense positivity for expression of Bcl-2 and FLIP were TUNEL negative. In addition, the number of neutrophils in the synovial lining and pannus significantly decreased from day 28 to day 35, suggesting that the cells undergoing apoptosis were neutrophils. Furthermore, at day 42 when TUNEL-positive cells were absent, Bcl-2 expression was diminished, while FLIP remained highly expressed in the pannus. CONCLUSION: The overall percentage of TUNEL-positive cells in the ankle was <1% except on days 28 and 35 post-adjuvant injection, suggesting that in AIA, similar to rheumatoid arthritis, a lack of apoptosis may contribute to disease progression. Furthermore, Bcl-2 and FLIP are temporally and differentially expressed during the pathogenesis of AIA. Inhibition of these molecules may augment synovial apoptosis and ameliorate the disease.


Subject(s)
Apoptosis/genetics , Arthritis, Experimental/physiopathology , Carrier Proteins/genetics , Intracellular Signaling Peptides and Proteins , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/physiopathology , CASP8 and FADD-Like Apoptosis Regulating Protein , Disease Models, Animal , Female , Gene Expression/physiology , In Situ Nick-End Labeling , Rats , Rats, Inbred Lew , Synovial Membrane/pathology
10.
J Immunol ; 165(12): 7199-206, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11120852

ABSTRACT

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-kappaB, C/EBPbeta, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IkappaBalpha, or dominant-negative versions of C/EBPbeta or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-kappaB activation significantly reduced the spontaneous and IL-1beta-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1ss-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPbeta modestly reduced constitutive and IL-1beta-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-kappaB, C/EBPbeta, and c-Jun were constitutively activated in RA FLS, but only NF-kappaB and c-Jun activity increased after IL-1beta. The reduction of cytokines by IkappaBalpha was mediated through inhibition of NF-kappaB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-kappaB was essential for IL-6 expression, because fibroblasts in which both NF-kappaB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-kappaB for the regulation of the constitutive and IL-1beta-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-kappaB as a therapeutic goal in RA.


Subject(s)
Arthritis, Rheumatoid/immunology , CCAAT-Enhancer-Binding Proteins/physiology , Fibroblasts/immunology , Fibroblasts/metabolism , I-kappa B Proteins , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , NF-kappa B/physiology , Proto-Oncogene Proteins c-jun/physiology , Synovial Membrane/immunology , Adenoviruses, Human/genetics , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , CCAAT-Enhancer-Binding Proteins/biosynthesis , CCAAT-Enhancer-Binding Proteins/metabolism , Cells, Cultured , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Dose-Response Relationship, Immunologic , Genetic Vectors/immunology , Humans , Interleukin-1/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-8/antagonists & inhibitors , Interleukin-8/genetics , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Proto-Oncogene Proteins c-jun/biosynthesis , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Skin/cytology , Skin/immunology , Skin/metabolism , Synovial Membrane/metabolism , Synovial Membrane/pathology , Transcription Factor AP-1/biosynthesis , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcriptional Activation/immunology
11.
Mol Cell Biol ; 20(23): 8855-65, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11073986

ABSTRACT

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.


Subject(s)
Apoptosis , DNA-Binding Proteins/biosynthesis , Homeodomain Proteins , I-kappa B Proteins , Macrophages/physiology , Mitochondria/physiology , NF-kappa B/metabolism , Proto-Oncogene Proteins c-bcl-2 , Repressor Proteins , Saccharomyces cerevisiae Proteins , Caspase 9 , Caspases/metabolism , Enzyme Activation , Homeostasis , Humans , Intracellular Membranes/physiology , Minor Histocompatibility Antigens , NF-KappaB Inhibitor alpha , Proton-Motive Force , Pyrrolidines/pharmacology , Replication Protein C , Thiocarbamates/pharmacology
12.
J Immunol ; 164(10): 5227-35, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10799883

ABSTRACT

The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.


Subject(s)
Fibroblasts/metabolism , Homeostasis , Mitochondria/physiology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Synovial Membrane/metabolism , Adenoviruses, Human/genetics , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/genetics , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Caspase 3 , Caspase Inhibitors , Cell Count , Cell Survival/genetics , Cells, Cultured , Down-Regulation/genetics , Enzyme Activation , Fibroblasts/pathology , Fibroblasts/physiology , Genetic Vectors/pharmacology , Homeostasis/genetics , Humans , Intracellular Membranes/physiology , Mitochondria/pathology , Osteoarthritis/metabolism , Osteoarthritis/pathology , Permeability , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/physiology , RNA, Catalytic/genetics , Synovial Membrane/pathology , Synovial Membrane/physiology , bcl-X Protein
13.
Nature ; 403(6767): 328-32, 2000 Jan 20.
Article in English | MEDLINE | ID: mdl-10659854

ABSTRACT

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.


Subject(s)
Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Dihydroorotase/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Multienzyme Complexes/metabolism , Allosteric Regulation , Amino Acid Sequence , Animals , Aspartate Carbamoyltransferase/antagonists & inhibitors , Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/antagonists & inhibitors , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cell Line , Cricetinae , Dihydroorotase/antagonists & inhibitors , Dihydroorotase/chemistry , Dihydroorotase/genetics , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Mesocricetus , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Molecular Sequence Data , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutagenesis, Site-Directed , Phosphoribosyl Pyrophosphate/metabolism , Phosphorylation , Pyrimidine Nucleotides/biosynthesis , Rats , Uridine Triphosphate/metabolism
14.
J Immunol ; 164(8): 4277-85, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10754326

ABSTRACT

The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-kappa B family members interact to regulate the human TNF-alpha gene. A 120-bp TNF-alpha promoter-reporter, possessing binding sites for NF-kappa B (kappa B3), C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-kappa B p65, and c-Jun, but not C/EBP beta, suppressed (p < 0.05-0.001) LPS-induced TNF-alpha secretion in primary human macrophages. Following LPS stimulation, NF-kappa B p50/p65 heterodimers bound to the kappa B3 site and c-Jun to the -103 AP-1 site of the TNF-alpha promoter. By transient transfection, NF-kappa B p65 and p50 synergistically activated the TNF-alpha promoter. In contrast, no synergy was observed between NF-kappa B p65, with or without NF-kappa B p50, and c-Jun or C/EBP beta, even in the presence of the coactivator p300. The contribution of the upstream kappa B binding sites was also examined. Following LPS stimulation, the kappa B1 site bound both NF-kappa B p50/p65 heterodimers and p50 homodimers. The binding by NF-kappa B p50 homodimers to the kappa B1, but not to the kappa 3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent kappa B3 and AP-1 sites in the human TNF-alpha promoter contribute independently to LPS-induced activation. Although both the kappa B1 and kappa B3 sites bound transcriptionally active NF-kappa B p50/p65 heterodimers, only the kappa B1 site contributed to down-regulation by NF-kappa B p50 homodimers.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation/immunology , Macrophages/metabolism , NF-kappa B/physiology , Nuclear Proteins/physiology , Proto-Oncogene Proteins c-jun/physiology , Tumor Necrosis Factor-alpha/genetics , Animals , CCAAT-Enhancer-Binding Proteins , Cell Line , Humans , Immune Tolerance/genetics , Immune Tolerance/immunology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Macrophages/immunology , Mice , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , NF-kappa B p50 Subunit , Promoter Regions, Genetic/immunology , Protein Binding/genetics , Protein Binding/immunology , Proto-Oncogene Proteins c-jun/antagonists & inhibitors , Transcription Factor RelA , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism
15.
J Exp Med ; 190(11): 1679-88, 1999 Dec 06.
Article in English | MEDLINE | ID: mdl-10587358

ABSTRACT

Macrophages differentiated from circulating peripheral blood monocytes are essential for host immune responses and have been implicated in the pathogenesis of rheumatoid arthritis and atherosclerosis. In contrast to monocytes, macrophages are resistant to Fas-induced cell death by an unknown mechanism. FLICE (Fas-associated death domain-like interleukin 1beta-converting enzyme)-inhibitory protein (Flip), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Here, we show that monocyte differentiation into macrophages was associated with upregulation of Flip and a decrease in Fas-mediated apoptosis. Overexpression of Flip protected monocytes from Fas-mediated apoptosis, whereas acute Flip inhibition in macrophages induced apoptosis. Addition of an antagonistic Fas ligand antibody to Flip antisense-treated macrophages rescued cultures from apoptosis, demonstrating that endogenous Flip blocked Fas-induced cell death. Thus, the expression of Flip in macrophages conferred resistance to Fas-mediated apoptosis, which may contribute to the development of inflammatory disease.


Subject(s)
Apoptosis , Carrier Proteins/genetics , Carrier Proteins/metabolism , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins , Macrophages/physiology , Monocytes/physiology , fas Receptor/physiology , CASP8 and FADD-Like Apoptosis Regulating Protein , Cell Differentiation , Cell Nucleus/ultrastructure , Cells, Cultured , Fas Ligand Protein , Humans , In Situ Nick-End Labeling , Macrophages/cytology , Membrane Glycoproteins/analysis , Monocytes/cytology , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transfection
16.
Clin Immunol ; 91(3): 271-82, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10370372

ABSTRACT

Rheumatoid arthritis synovial tissue was examined and compared with osteoarthritis tissue for the presence of the nuclear transcription factor C/EBP beta (NF-IL-6). The region (lining or sublining), cell type, and subcellular distribution (cytoplasmic or nuclear) of the expression of C/EBP beta was characterized. Rheumatoid arthritis synovial fluid and blood and normal peripheral blood were also examined. C/EBP beta was detected in the synovial lining and in sublining cells of synovial tissue from patients with both rheumatoid and osteoarthritis. A significant (P < 0.001 and < 0.05, respectively) increase in the percentage of cells with nuclear staining was seen in the lining layer, compared to cells in the sublining region, in rheumatoid and osteoarthritis. In both diseases a strong correlation (r = 0.79, P < 0.001) was observed between the percentage of cells in the synovial lining that were positive for nuclear C/EBP beta and lining cell depth. Two-color immunohistochemistry demonstrated that both macrophages and fibroblast-like synoviocytes were positive for nuclear C/EBP beta. The presence of C/EBP beta was confirmed by immunohistochemistry and Western blot analysis with isolated synovial fibroblasts. Nuclear C/EBP beta was also detected in rheumatoid synovial fluid monocytes/macrophages, but not in lymphocytes or neutrophils. Western blot analysis confirmed the presence of C/EBP beta in these cells. The intensity of C/EBP beta staining was greater (P < 0.001) in synovial fluid monocytes than in those from normal or rheumatoid peripheral blood. In conclusion, the enhanced nuclear staining for C/EBP beta in the synovial lining, compared to the sublining, suggesting activation in the lining, and the positive correlation of lining layer depth with the percentage of cells in the lining positive for nuclear C/EBP beta, suggest a potential role for C/EBP beta in chronic inflammation. The regulation of the production or activity of C/EBP beta, to inhibit inflammatory mediator expression by synovial macrophages and fibroblasts, offers a novel approach to therapeutic intervention.


Subject(s)
Arthritis, Rheumatoid/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Arthritis, Rheumatoid/immunology , CCAAT-Enhancer-Binding Proteins , Cell Nucleus/metabolism , Fibroblasts/immunology , Fibroblasts/metabolism , Humans , Immunohistochemistry , Inflammation/immunology , Inflammation/metabolism , Macrophage Activation , Macrophages/immunology , Macrophages/metabolism , Monocytes/immunology , Monocytes/metabolism , Osteoarthritis/immunology , Osteoarthritis/metabolism , Synovial Membrane/immunology , Synovial Membrane/metabolism
17.
Appl Opt ; 36(33): 8710-23, 1997 Nov 20.
Article in English | MEDLINE | ID: mdl-18264420

ABSTRACT

Definitive data on the absorption spectrum of pure water from 380 to 700 nm have been obtained with an integrating cavity technique. The results are in good agreement with those recently obtained by our group with a completely independent photothermal technique. As before, we find that the absorption in the blue is significantly lower than had previously been generally believed and that the absorption minimum is at a significantly shorter wavelength, i.e., 0.0044 ? 0.0006 m(-1) at 418 nm. Several spectroscopic features have been identified in the visible spectrum to our knowledge for the first time.

18.
Arthritis Rheum ; 39(9): 1566-75, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8814069

ABSTRACT

OBJECTIVE: Angiogenesis is an integral component of the vasculoproliferative phase of rheumatoid arthritis (RA). Recently, a heparin-binding cytokine termed hepatocyte growth factor (HGF), or scatter factor (due to its ability to disperse cohesive epithelial colonies), was described. We conducted this study to investigate the hypothesis that this cytokine was present in the milieu of the inflamed joint, and that it contributed to the chemotaxis of endothelial cells in the synovial tissue. METHODS: We examined synovial fluid, synovial tissue, and peripheral blood from 91 patients with RA and other arthritides. We used 83 total samples in an enzyme-linked immunosorbent assay to quantitate the HGF in synovial fluids and peripheral blood. To determine whether the HGF was biologically active, an epithelial scatter factor assay was performed. Immunohistochemical analysis was used to determine localization in synovial tissues. To define a function for synovial HGF, we preincubated rheumatoid synovial fluids with neutralizing anti-HGF and measured the ability of these synovial fluids to induce endothelial chemotaxis. RESULTS: Synovial fluid from patients with RA contained a mean +/- SEM HGF concentration of 2.0 +/- 0.3 ng/ml, while synovial fluid from patients with other arthritides (including inflammatory arthritis) contained 2.4 +/- 0.7 ng/ml HGF. Osteoarthritis (OA) patient samples contained the smallest quantities of synovial fluid HGF at 0.9 +/- 0.1 ng/ml. RA synovial fluid contained significantly more HGF than did RA peripheral blood (1.1 +/- 0.2 ng/ml) (P < 0.05). Rheumatoid synovial fluids induced more scattering of cells than did OA synovial fluids, suggesting a role for this cytokine in rheumatoid joint destruction. Interleukin-1 beta induced expression of rheumatoid synovial tissue fibroblast antigenic HGF and scatter factor activity. Immunohistochemically, HGF, as well as the HGF receptor (the met gene product), localized to significantly more rheumatoid synovial tissue lining cells than normal lining cells (P < 0.05). Both HGF and its receptor immunolocalized to subsynovial macrophages as well. Levels of synovial tissue immunoreactive HGF correlated positively with the number of synovial tissue blood vessels. Anti-HGF neutralized a mean of 24% of the chemotactic activity for endothelial cells found in 10 rheumatoid synovial fluid samples. CONCLUSION: These results indicate that synovial HGF may contribute to the vasculoproliferative phase of inflammatory arthritides such as RA, by inducing HGF-mediated synovial neovascularization. These findings point to a newly described role for HGF in the fibroproliferative phase of RA-associated synovitis.


Subject(s)
Arthritis, Rheumatoid/blood , Hepatocyte Growth Factor/analysis , Hepatocyte Growth Factor/physiology , Osteoarthritis/blood , Receptor Protein-Tyrosine Kinases/analysis , Synovial Fluid/chemistry , Synovial Membrane/chemistry , Chemotaxis , Endothelium, Vascular/pathology , Fibroblasts/metabolism , Hepatocyte Growth Factor/blood , Humans , Proto-Oncogene Proteins c-met , Synovial Fluid/metabolism , Synovial Membrane/metabolism , Synovial Membrane/pathology
19.
Arthritis Rheum ; 39(9): 1576-87, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8814070

ABSTRACT

OBJECTIVE: To determine the expression of matrix metalloproteinase 9/gelatinase B (MMP-9) in synovial fluid (SF), plasma, and synovial tissue from individuals with rheumatoid arthritis (RA), inflammatory arthritis (IA), and osteoarthritis (OA), using specific monoclonal antibody reagents. METHODS: Gelatinolytic activity in the SF and plasma of patients with RA, IA, and OA was assessed by gelatin zymography. A mouse monoclonal antiserum, 277.13, which selectively recognizes soluble latent forms of human MMP-9, was used to quantitate MMP-9 levels in patient synovial effusions, plasma, and synovial tissue with a capture sandwich enzyme-linked immunosorbent assay (ELISA). Fifty-one SF samples (31 RA, 9 OA, 11 IA) were analyzed. Immunolocalization of MMP-9 in RA, OA, and normal synovium was investigated using MMP-9-specific antisera. RESULTS: MMP-9 antigen levels in synovial effusions were elevated 67-fold in RA samples compared with OA samples. In addition, although MMP-9 antigen levels in IA synovial effusions were 2.7-fold less than the values in RA samples, they were elevated 34-fold over the values in OA samples. These data indicate an association between increased MMP-9 levels and inflammatory arthritis. A predominant 92-kd gelatinolytic activity (specifically inhibited by EDTA) was evident in RA and IA samples, but no activity was observed in OA samples. Among 86 plasma samples (17 RA, 9 IA, 60 normal controls) analyzed for MMP-9 antigen levels by immunocapture ELISA, MMP-9 antigen levels were elevated 7-fold in RA plasma compared with normal plasma. RA synovial tissue extracts demonstrated elevated levels of MMP-9 antigen compared with OA synovial tissue. MMP-9 immunolocalization studies demonstrated expression in infiltrating leukocytes (neutrophils and macrophages), endothelial cells, and synovial fibroblasts in RA synovium. CONCLUSION: Latent MMP-9 and/or MMP-9-tissue inhibitor of metalloproteinases 1 (TIMP-1) complexes are elevated in RA and IA SF compared with OA SF. In addition, MMP-9 is increased in RA plasma versus normal control plasma. Synovial tissue levels of MMP-9 antigen are also elevated in RA versus OA. The tissue distribution of MMP-9 within RA synovium is localized to sites of inflammation comprising surface synovial lining cells, endothelium, and leukocytes. Taken together, these observations suggest that connective tissue turnover occurs as a result of excessive MMP activity over TIMP action in the invading pannus, periarticular tissue, or SF. Further studies such as those used in the present investigation will help elucidate the role of a number of different enzymes and inhibitors in the destructive arthropathies.


Subject(s)
Arthritis, Rheumatoid/enzymology , Collagenases/metabolism , Osteoarthritis/enzymology , Synovial Fluid/enzymology , Synovial Membrane/enzymology , Adult , Arthritis/blood , Arthritis/enzymology , Arthritis, Rheumatoid/blood , Collagenases/analysis , Collagenases/blood , Female , Humans , Male , Matrix Metalloproteinase 9 , Middle Aged , Osteoarthritis/blood
20.
Clin Immunol Immunopathol ; 78(3): 276-82, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8605703

ABSTRACT

A number of adhesion molecules have been identified in synovial tissues of patients with rheumatoid arthritis (RA). Some of them are upregulated and may play an important role in the inflammatory processes of the diseased joint. In addition to synovial tissue cell surface expression, synovial fluids contain soluble forms of many adhesion molecules, such as intercellular adhesion molecule-1, E-selectin (sE-selectin), and L-selectin. In this study, we investigated the expression of soluble P-selectin (sP-selectin) and intercellular adhesion molecule-3 (sICAM-3) in synovial fluids from patients with RA, osteoarthritis (OA), and other forms of arthritis. sP-selectin and sICAM-3 levels in RA synovial fluids were significantly increased compared to those in OA. The levels of sP-selectin++ in synovial fluids correlated with sICAM-3 and sE-selectin in synovial fluids. The levels of sICAM-3 in synovial fluids correlated with synovial fluid leukocyte counts and erythrocyte sedimentation rate. In vitro, synovial fluid mononuclear cells produced sICAM-3 spontaneously. Elevated levels of sP-selectin and sICAM-3 in RA synovial fluids compared to OA may indicate inflammatory interactions between endothelial cells, leukocytes, and other synovial cells in the diseased joint.


Subject(s)
Antigens, CD , Antigens, Differentiation , Arthritis , Cell Adhesion Molecules/analysis , P-Selectin/analysis , Synovial Fluid/chemistry , Arthritis/classification , Blood Sedimentation , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Humans , Leukocyte Count , Leukocytes, Mononuclear , Neutrophils , Solubility , Synovial Fluid/cytology
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