Lysophosphatidic acid mediates migration of human mesenchymal stem cells stimulated by synovial fluid of patients with rheumatoid arthritis

Migration of mesenchymal stem cells plays a key role in regeneration of injured tissues. Rheumatoid arthritis (RA) is a chronic inflammatory disease and synovial fluid (SF) reportedly contains a variety of chemotactic factors. This study was undertaken to investigate the role of SF in migration of human bone marrow-derived mesenchymal stem cells (hBMSCs) and the molecular mechanism of SF-induced cell migration. SF from RA patients greatly stimulated migration of hBMSCs and the SF-induced migration was completely abrogated by pretreatment of the cells with the lysophosphatidic acid (LPA) receptor antagonist Ki16425 and by small interfering RNA- or lentiviral small hairpin RNA-mediated silencing of endogenous LPA₁/Edg2. Moreover, SF from RA patients contains higher concentrations of LPA and an LPA-producing enzyme autotoxin than normal SF. In addition, SF from RA patients increased the intracellular concentration of calcium through a Ki16425-sensitive mechanism and pretreatment of the cells with the calmodulin inhibitor W7 or calmodulin-dependent protein kinase II inhibitor KN93 abrogated the SF-induced cell migration. These results suggest that LPA-LPA₁ plays a key role in the migration of hBMSCs induced by SF from RA patients through LPA₁-dependent activation of calmodulin-dependent protein kinase II.     

Elevated neutrophil respiratory burst activity in essential hypertensive patients

Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients’ neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control’s neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication.     

Isolation and properties of oxidized alpha-1-proteinase inhibitor from human rheumatoid synovial fluid

Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P₁ reactive site and the other at P₈, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed $\text{in vivo}$, presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur.     

Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid.

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium.     

Annexin-1 mediates TNF-alpha-stimulated matrix metalloproteinase secretion from rheumatoid arthritis synovial fibroblasts

Annexins are intracellular molecules implicated in the down-regulation of inflammation. Recently, annexin-1 has also been identified as a secreted molecule, suggesting it may have more complex effects on inflammation than previously appreciated. We studied the role of annexin-1 in mediating MMP-1 secretion from rheumatoid arthritis (RA) synovial fibroblasts (SF) stimulated with TNF-alpha. TNF-alpha induced a biphasic secretion of annexin-1 from RA SF. Early (< or = 60 min), cycloheximide-independent secretion from preformed intracellular pools was followed by late (24 h) cycloheximide-inhibitable secretion requiring new protein synthesis. Exogenous annexin-1 N-terminal peptide Ac2-26 stimulated MMP-1 secretion in a dose- (EC(50) approximately 25 microM) and time- (8-24 h) dependent manner; full-length annexin-1 had a similar effect. Down-regulation of annexin-1 using small interfering RNA resulted in decreased secretion of both annexin-1 and MMP-1, confirming that annexin-1 mediates TNF-alpha-stimulated MMP-1 secretion. Erk, Jnk, and NF-kappaB have been implicated in MMP-1 secretion. Erk, Jnk, and NF-kappaB inhibitors had no effect on annexin-1 secretion stimulated by TNF-alpha but inhibited MMP-1 secretion in response to Ac2-26, indicating that these molecules signal downstream of annexin-1. Annexin-1 stimulation of MMP-1 secretion was inhibited by both a formyl peptide receptor antagonist and pertussis toxin, suggesting that secreted annexin-1 acts via formyl peptide family receptors, most likely FPLR-1. In contrast to its commonly appreciated anti-inflammatory roles, our data indicate that annexin-1 is secreted by RA SF in response to TNF-alpha and acts in an autacoid manner to engage FPRL-1, activate Erk, Jnk, and NF-kappaB, and stimulate MMP-1 secretion.     

Activation of the human neutrophil respiratory burst with zymosan-activated serum

Zymosan-activated serum (ZAS) stimulated a time- and concentration-dependent generation of superoxide anion (O₂^?) by human neutrophils. O₂^? production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS. O₂^? generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS. The amount of O₂^? produced by ZAS stimulated neutrophils was enhanced in the presence of extracellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS-elicited O₂^? production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O₂^? in response to 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB₄), demonstrates the stimulus specific nature of ZAS-induced desensitization. Thus, ZAS, which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represénts a relevent probe for investigating neutrophil function.     

Immunological detection of myeloperoxidase in synovial fluid from patients with rheumatoid arthritis.

We have used rocket immunoelectrophoresis and immunoblotting to detect myeloperoxidase in synovial fluid from patients with rheumatoid arthritis. This protein was enzymatically inactive but its identity as myeloperoxidase was confirmed by comparing its subunit structure with that of the purified enzyme. When neutrophils were stimulated to secrete myeloperoxidase in vitro, a polypeptide with an apparent molecular mass of 62 kDa was detected extracellularly by immunoblotting. Neutrophils isolated from synovial fluid showed a reduced level of this 62 kDa polypeptide but it was detected extracellularly in synovial fluid by immunoblotting. Thus, we conclude that neutrophils in synovial fluid from patients with rheumatoid arthritis have been activated in vivo to secrete myeloperoxidase and propose that the products of this enzyme system can contribute to the tissue damage associated with this disease.     

Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.     

Evidence for oxidised low density lipoprotein in synovial fluid from rheumatoid arthritis patients

The oxidative modification of human LDL has been implicated in atherosclerosis, but the mechanisms by which such modification occurs in vivo are not fully understood. In the present study, we have isolated LDL from knee-joint synovial fluid of patients with rheumatoid arthritis. We demonstrate that such LDL is oxidatively modified as evidenced by an increased negative charge, distorted particulate nature and more rapid degradation by cultured macrophages. These results indicate that formation of oxidised LDL is associated with the local inflammatory response. Because the cellular interactions in rheumatoid arthritis have analogies with those in atherogenesis, we suggest that the rheumatoid joint is a useful model of atherosclerosis in which the in vivo process of LDL oxidation may be readily studied.     

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).     

Purification of granulocyte neutral protease from human blood and rheumatoid synovial fluid.

The neutral protease activity of human synovial fluid cells, like that of peripheral blood leucocytes, is located in a granule fraction. It can be solubilised by various agents but only 1 M neutral salts do so without inactivation. Salt-solubilised neutral protease has been purified (300 X) from synovial fluid cells; like preparations obtained in the same way (600 X purified) from peripheral blood leucocytes, it has a broad pH profile of activity (pH 7--10.5) and in this, as well as in substrate specificity and sensitivity to activators and inhibitors, it behaves as a serine-histidine type protease similar to elastase (EC 3.4.21.11). The product showed two major components on polyacrylamide gel electrophoresis. Collagenase or chymotrypsin-like activity were not detected.     

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.     

Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.     

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.     

Phosphatidylinositol-linked FcRIII mediates exocytosis of neutrophil granule proteins, but does not mediate initiation of the respiratory burst

In this report, we present data on the activation of different neutrophil effector functions by two distinct Fc-gamma receptors, FcRII and FcRIII. We and others have shown previously that IgG-dependent activation of phagocytosis and superoxide generation is mediated via FcRII. IgG-dependent exocytosis of granule proteins was assessed with Staphylococcus aureus Oxford opsonized with human IgG or with IgG-coated latex. Both anti-FcRII mAb and anti-FcRIII-F(ab')2 mAb inhibited this release, whereas the combination of these mAb inhibited this process more strongly than either mAb alone. This indicates that both FcRII and FcRIII are involved in IgG-dependent release of granule proteins. Cross-linking of the receptors by anti-FcR mAb and F(ab')2 fragments of goat-anti-mouse-Ig showed again that both FcRII and FcRIII mediate lysozyme release, whereas cross-linking of a control antigen (CD67) did not. By measuring the release of elastase and lactoferrin, we found that cross-linking of either FcRII or FcRIII induced release of both azurophilic and specific granules. Under these conditions, we did not measure any activation of the respiratory burst. When FcRIII was removed by treatment of neutrophils with glycosylphosphatidylinositol-specific phospholipase C, the lysozyme release induced by cross-linking of FcRIII was lower than the release from control neutrophils, whereas the release induced by cross-linking of FcRII was similar. Therefore, we conclude that IgG-dependent activation of neutrophils follows two distinct pathways: one via transmembrane FcRII, activating both the NADPH oxidase and the release of granule proteins (as was demonstrated previously by us and by others), and the other via phosphatidylinositol-linked FcRIII, activating exocytosis of granule proteins.