Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis

Extracellular phospholipase A2 was purified about 1.7 X 10(5) fold to near homogeneity from human synovial fluid of rheumatoid arthritis by sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl, and reversed-phase HPLC. The final preparation showed a single band on SDS-polyacrylamide gel electrophoresis, and its molecular mass was estimated to be approximately 13,700 daltons. The purified enzyme had a pH optimum of 9.0 and required Ca2+ for maximum activity. It hydrolyzed phosphatidyl-ethanolamine more effectively than phosphatidylserine and phosphatidylcholine. These properties were similar to those of an extracellular phospholipase A2 detected in the peritoneal cavity of caseinate-treated rats.     

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.     

Multiple forms of phospholipase A2 in arthritic synovial fluid

Phospholipase A2 (PLA2) has been purified to homogeneity from human arthritic synovial fluid. The activity resolved into multiple peaks by preparative HPLC. The most abundant peak (A) was present in synovial fluid from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. A second major peak (B) was variable and lower in relative abundance, but was distinguishable from peak A by its stimulated activity in the presence of either 0.5 M Tris or 0.1% sodium deoxycholate (DOC), in addition to its longer HPLC column retention time. Both peaks required Ca2+ and showed optimal activity in DOC/phosphatidylcholine (PC) mixed micelle assays between pH 8.0 and 9.0. Both peaks showed higher activity with PC as substrate than with PI, however peak A exhibited higher activity with PE than PC. Upon preparative SDS-polyacrylamide gel electrophoresis, both peaks of PLA2 activity were resolved as proteins of approximately 14,000 Da. The N-terminal sequence obtained from purified peak A material matched that of a recent similar isolate (Hara et al. (1988) J. Biochem. 104, 326-328).     

Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid

IgG Fc- cytotoxic cells found in the synovial fluid of patients with rheumatoid arthritis have natural killer (NK)-like characteristics but can kill NK-resistant cell lines as well. The phenotype of these cells was defined by complement-mediated lysis with monoclonal antibodies. The synovial fluid killer cell activity was significantly reduced by treatment with complement and OKT11 and 4F2, but the cytotoxic T cells did not express the NK-related antigens OKM1 and Leu-7, nor the cytotoxic T lymphocyte-specific antigen, OKT8. These results demonstrate that the synovial fluid killer cells resemble the activated T cells generated in an autologous mixed leukocyte reaction or in the treatment of peripheral blood mononuclear cells with interleukin 2, and they are distinct from the conventional NK cells found in blood.     

Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid

The amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predominant amino acids in the phospholipase A2 were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu- Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the "Group I" enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.     

Kallikrein in synovial fluid with rheumatoid arthritis

The levels of kallikrein and collagenase in synovial fluid from rheumatoid arthritis (RA) patients were examined and the role of kallikrein in procollagenase activation is discussed. Both prekallikrein and active kallikrein in synovial fluid from patients with RA were significantly elevated when compared to synovial fluid from patients with osteoarthritis (OA). In RA synovial fluid, the ratio of the active form to total kallikrein was also higher than that in OA synovial fluid. Both active collagenase and the alpha 2-macroglobulin (alpha 2M)-collagenase complex in RA synovial fluid were higher than in OA synovial fluid. A partial correlation (r = 0.58) between active kallikrein and total collagenase (active and alpha 2M-collagenase complex) was observed in RA synovial fluid. These observations indicate that both kallikrein and collagenase are associated with the destruction of cartilage, but the role of kallikrein in procollagenase activation was not fully clarified.     

Monoclonal antibodies against human synovial phospholipase A2

Four monoclonal antibodies (HP-1, HP-2, HP-3 and HP-4) with differing reactivities were raised against human synovial fluid phospholipase A2. None of them bound to exocrine phospholipases A2, such as those from pancreas or snake venom. However, antibodies HP-1 and HP-3 showed cross-reactivity with rabbit and rat platelet secretory phospholipases A2, which share common enzymatic and structural features with the human synovial enzyme. Antibodies HP-1, HP-2 and HP-3 inhibited the activity of human synovial phospholipase A2. The antibodies were used to develop a rapid immunoaffinity column chromatographic procedure for enzyme purification. In some preparations, the recovery of total activity after immunoaffinity column chromatography was more than 100% suggesting the existence of endogenous inhibitory factors of phospholipase A2 in human synovial fluid.     

Endothelial protein C receptor-associated invasiveness of rheumatoid synovial fibroblasts is likely driven by group V secretory phospholipase A2

Introduction

Rheumatoid synovial fibroblasts (RASFs) mediate joint inflammation and destruction in rheumatoid arthritis (RA). Endothelial protein C receptor (EPCR) is a specific receptor for the natural anticoagulant activated protein C (APC). It mediates the cytoprotective properties of APC and is expressed in rheumatoid synovial tissue. A recent report shows that group V secretory phospholipase A2 (sPLA₂V) prevents APC from binding to EPCR in endothelium and inhibits EPCR/APC function. The aim of this study was to investigate the expression and function of EPCR on RASFs.

Methods

Human synovial fibroblasts (SFs) were isolated from RA or osteoarthritis (OA) synovial tissues and treated with control, EPCR, or sPLA₂V small interfering RNA (siRNA); recombinant human APC, tumor necrosis factor-alpha (TNF-α), or sPLA₂V. RASF viability and migration/invasion were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and collagen gel migration/invasion assays, respectively, and cartilage degradation by 1,9-dimethylmethylene blue (DMMB) assay in the presence of human OA articular cartilage explants. The expression or activation of cytokines, EPCR, cadherin-11, mitogen-activated protein (MAP) kinases, and nuclear factor-kappa-B (NF-κB) or both were detected by enzyme-linked immunosorbent assay, Western blotting, or immunostaining.

Results

EPCR was expressed by both OASFs and RASFs but was markedly increased in RASFs. When EPCR was suppressed by siRNA or blocking antibody cell viability, cell invasion and cartilage degradation were reduced by more than 30%. Inflammatory mediators interleukin-1-beta (IL-1β), cadherin-11, and NF-κB were significantly reduced by EPCR suppression under control or TNF-α-stimulated conditions. The expression or activation (or both) of MAP kinases ERK, p38, and JNK were also markedly decreased in cells transfected with EPCR siRNA. Further analysis revealed that sPLA₂V co-localized with EPCR on RASFs. Suppression of sPLA₂V reduced cell viability and cartilage degradation and increased APC binding to RASFs. Conversely, recombinant sPLA₂V increased cartilage degradation, blocked APC binding to RASFs, and could not rescue the effects induced by EPCR suppression.

Conclusions

Our results demonstrate that EPCR is overexpressed by RASFs and mediates the aggressive behavior of RASFs. This function of EPCR is contrary to its cytoprotective role in other settings and is likely driven by sPLA₂V.     

A latent collagenase from rheumatoid synovial fluid. Purification and partial characterization

1. A latent collagenase (EC 3.4.24.3) has been isolated from rheumatoid synovial fluids and purified by (NH4)2SO4 precipitation and column chromatography, utilising Sephadex G-150, DEAE Sephadex A-50 and Sephadex G-100 superfine grade. 2. The final preparation activated by trypsin (EC 3.4.21.4) had a specific activity against thermally reconstituted collagen fibrils of 259 micrograms collagen degraded/min per mg enzyme protein, representing a nearly 800-fold increase over that of the original rheumatoid synovial fluid. 3. The latent collagenase preparation can be activated by trypsin and to some extent by HgCl2 but not by 3 M NaSCN, 3.5 M NaCl, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or p-chloromercuribenzoate. 4. Inhibition studies and the acrylamide gel electrophoretic pattern of collagen degradation products showed that the trypsin-activated enzyme has the essential features of a neutral collagenase. 5. The molecular weights, determined by calibrated gel filtration, were 52 000 and 43 000 for the latent and the activated enzyme, respectively. 6. The nature of the latency of synovial fluid collagenase is discussed.     

Phospholipid synthesis by extracellular phospholipase A2 in organic solvents

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.     

Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis

We measured and compared the activities of various kinds of proteinases, such as cysteine, serine, aspartic, and metalloproteinases, in synovial fluids of 16 patients with rheumatoid arthritis (RA) and 18 patients with osteoarthritis (OA). More than 19-fold higher activity of cathepsin B and about 6-fold higher activity of prolylendopeptidase, compared to those of OA, were accumulated in RA fluid. Moreover, levels of cathepsins B and S using the corresponding sandwich enzyme immunoassays were statistically higher in RA fluid than those in OA. Significant amounts of 41-kDa and 35-kDa procathepsin L were detected in RA fluid using gelatin zymography, while 41-kDa enzyme alone was detected in OA. Cathepsin B in RA fluid could degrade collagen, and this degradation was suppressed by the addition of CA-074, a specific inhibitor of cathepsin B. Therefore, cathepsin B may participate in joint destruction of RA, and its inhibitor may be effective for RA care.     

The influence of synovial fluid on lymphocyte functions in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic recurrent and systemic inflammatory disease affecting around 1% of the population, that primarily involves the joints. In this study, we determined the Th1/Th2 lymphocytes ratio at the site of rheumatoid inflammation and the influence of the synovial fluid (SF) on the secretory and proliferative function in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC), obtained from patients with RA. Our results showed significant differences concerning the mononuclear cells and the CD4/CD8 ratio in synovial fluid and peripheral blood of patients. In SF prevailed Th1 cells, while in peripheral blood we found another cytokine profile of T lymphocytes. Also synovial fluid lymphocytes had a low PHA-stimulated blastogenic response. Patients plasma and synovial fluid showed an inhibitory effect on prolipheration indexes.     

Stimulatory role of lysophosphatidic acid in cyclooxygenase-2 induction by synovial fluid of patients with rheumatoid arthritis in fibroblast-like synovial cells

While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E(2) production, in RA synovial cells. The synovial fluid-induced actions were inhibited by G(i/o) protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1alpha- or IL-1beta-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA(1) receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.     

Cytochemical investigation of cathepsin B in rheumatoid synovial membrane and fluid

Cathepsin B was cytochemically investigated in the cells of synovial membranes and in the cell pellet of synovial fluids obtained from 50 patients with rheumatoid arthritis and eight patients with various nonrheumatoid arthropathies. The activity of Cathepsin B was estimated by using the substrate N-alpha-benzoyl-DL-arginine-naphthylamide HCl and diazoic dye Fast Corinth V in phosphate buffer pH 6.0 in the presence of EDTA and cysteine. A significant activity of cathepsin B was shown in lining mesothelial cells, in macrophages of the submesothelial infiltrations, as well as in fibroblasts prominent in the deep areas of rheumatoid synovial membranes. In the cell pellets of synovial fluids the highest activity of cathepsin B was found in the macrophages and polymorphonuclear leukocytes, accompanied by a variable activity in lymphocytes. The considerable activity of cathepsin B, an enzyme with degradative action upon collagen and proteoglycans, in the main cellular populations of rheumatoid synovial membranes and fluids, suggests its involvement in the genesis and maintenance of rheumatoid lesions.     

Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.

Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.     

Characterization of phospholipase A2 of mycoplasma species

As phospholipases of mycoplasma species may play a role in the pathogenesis of respiratory tract and urogenital tract diseasesMycoplasma mycoides andAcholeplasma laidlawii were examined as to the production of phospholipase A₂ (PLA) and attempts were made to purify and characterize it. Both species produced PLA. The purified enzyme was found to be heat-labile, active at alkaline pH, revealing a single band in polyacrylamide gel electrophoresis. Metal ions such as calcium and barium, increased its activity whereas solvents at high concentrations decreased it. It was resistant to surfactants.