Myeloperoxidase and its products in synovial fluid of patients with treated or untreated rheumatoid arthritis.

OBJECTIVE: Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), interleukin (IL)-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. METHODS: Patients (n = 105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. Disease activity score (DAS-28) was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine (Cl-Tyr), homocitrulline (Hcit), IL-8, and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. RESULTS: DAS-28 and CRP levels were not significantly different between groups. MPO activity, and MPO, Cl-Tyr, and Hcit levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. CONCLUSIONS: MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and Hcit in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations.

Antigens and granularity of blood monocytes in relation to inflammatory markers and lipids in postmenopausal women.

OBJECTIVES: The menopause is associated with an increase of inflammatory markers (C-reactive protein, fibrinogen), cytokines (INFgamma, TNF, etc.) and blood lipoproteins. In vitro, CRP, LDL and fibrinogen can modulate or potentiate interleukines production by monocytes. The aim of this work was to study, the relationships in vivo between hs-CRP, fibrinogen, lipoproteins and the phenotype of circulating monocytes. METHODS: The monocytes phenotype, in postmenopausal women (n=26) without history of cardiovascular disease, was determined, by flow cytometry, measuring granularity and CD14, HLA-DR and CD62-L antigens expression. Blood monocytes were divided in CD14+dim monocytes (low CD14 expression) and CD14+bright monocytes (high CD14 expression). RESULTS: HLA-DR was negatively correlated with hs-CRP and fibrinogen. The relationships between ApoB, LDL/ApoB ratio and CD14 expression was restricted to the CD14+bright monocytes. Blood lipids, i.e. total cholesterol, LDL-c and ApoB were correlated with the granularity of both subsets. CD14+dim monocytes were characterized by a low granularity and CD62-L expression. CONCLUSIONS: Our data show that fibrinogen and hs-CRP are correlated with a reduced antigen-presenting capacity. Expression of CD14 on CD14+bright monocytes is negatively associated to atherogenic LDL. Blood monocytes granularity was positively correlated with serum lipids indicating that monocytes could uptake modified LDL in circulation and not restricted to subendothelial space.

Oxidation of low density lipoproteins by myeloperoxidase at the surface of endothelial cells: an additional mechanism to subendothelium oxidation.

The present paradigm of atherogenesis proposes that low density lipoproteins (LDL) are trapped in subendothelial space of the vascular wall where they are oxidized. Myeloperoxidase (MPO) plays a key role in oxidative damage. We propose that LDL oxidation by myeloperoxidase (Mox-LDL) could occur at the surface of the endothelial cells and not restricted to the subendothelial space. The triad constituted by endothelial cells, circulating LDL and MPO in close interaction, constitutes a synergic mechanism for the genesis of Mox-LDL.

Monoclonal antibodies against LDL progressively oxidized by myeloperoxidase react with ApoB-100 protein moiety and human atherosclerotic lesions.

Oxidized-LDL are involved in atherosclerosis pathogenesis, while the production of anti-ox-LDL monoclonal antibodies is critical for the development of diagnostic tools. This work reports the production of four monoclonal antibodies raised against human LDL, oxidized at different levels by the myeloperoxidase system. Characterization of these monoclonal antibodies showed that they do not cross-react with neither native LDL, VLDL nor hydrogen peroxide or Cu(2+)-oxidized LDL. Three of these antibodies recognize an epitope restricted to the protein moiety of mildly oxidized LDL, whereas the fourth antibody was partly dependent on the lipid presence of strongly oxidized LDL. All the antibodies were shown to react with human atherosclerotic lesions.

Human recombinant myeloperoxidase antiviral activity on cytomegalovirus.

In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively.

Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase.

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.

A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase.

Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9 x 10(7) M(-1) s(-1), pH 7 and 15 degrees C) and reduction of compound I to compound II (3.0 x 10(4) M(-1) s(-1), pH 7 and 15 degrees C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6 x 10(4) M(-1) s(-1), 1.4 x 10(6) M(-1) s(-1), 1.4 x 10(7) M(-1) s(-1) and 1.4 x 10(7) M(-1) s(-1), respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3 x 10(5) M(-1) s(-1)) and of compound II back to the resting enzyme (5.0 x 10(3) M(-1) s(-1)). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.

Native and recombinant proteins to analyze auto-antibodies to myeloperoxidase in pauci-immune crescentic glomerulonephritis.

The prevalence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) in pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is dependent on the assay(s) used. We investigated the frequency of MPO-ANCA as detected by different assays for MPO-ANCA in a large cohort of patients with biopsy-proven pauci-immune NCGN. Sera from 121 consecutive untreated patients presenting with pauci-immune NCGN were tested for ANCA directed to proteinase-3 (PR3) at diagnosis. PR3-ANCA negative sera were tested by direct ELISA using recombinant or native MPO and by capture ELISA using two different specific monoclonal antibodies directed to MPO and three different antigenic sources. Sera from 80 relevant disease controls were tested to explore the specificity of the different assays. Thirty-eight out of 121 patients (31%) with pauci-immune NCGN did not have PR3-ANCA. Sufficient amounts of serum from 30 of these 38 PR3-ANCA negative patients were available for further testing. Recombinant and native MPO were recognized by similar numbers of sera in a direct ELISA (recombinant MPO: 93%, native MPO: 93%) and a capture ELISA (recombinant MPO: 77-87%, native MPO: 93%). Sera of patients with PR3-ANCA positive pauci-immune NCGN and disease controls were less frequently positive for MPO-ANCA in a capture ELISA (recombinant MPO: 3-7%, native MPO: 6-7%) than in a direct ELISA (recombinant MPO: 25%, native MPO: 13%). Both direct and capture ELISA assays using either native or recombinant MPO are sensitive techniques to detect MPO-ANCA in patients with pauci-immune NCGN. A capture ELISA performs better than a direct ELISA because it combines a higher specificity with a comparable sensitivity. Recombinant MPO is a good alternative for native MPO when used as antigen in a capture ELISA, but not when used in a direct ELISA because of lower specificity in this latter assay.

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs investigated with a human recombinant enzyme.

Non-steroidal anti-inflammatory drugs (NSAIDs) were investigated for their ability to affect the chlorinating activity of human myeloperoxidase and to scavenge HOCl, the main myeloperoxidase system product. Fourteen drugs representative of various NSAIDs families were tested with the chlorination of taurine used as a detection system. All were unable to inhibit taurine chlorination in a system without myeloperoxidase. In contrast, most of them induced a dose-dependent inhibition of the taurine chlorination mediated by a myeloperoxidase/H2O2/Cl- system. This took place at variable drug concentrations and IC50 were calculated. The inhibitory effect was therefore due to a direct interaction with the enzyme rather than to HOCl scavenging. A spectroscopic method used to measure the myeloperoxidase compound II lifetime in presence of the different drugs showed that all the drugs, which inhibited chlorination activity were able to induce accumulation of compound II. The extent of chlorinating activity inhibition (IC50) was inversely related to the duration of the block of enzyme in compound II form. This further demonstrates that myeloperoxidase is an interesting target for anti-inflammatory therapy. The recombinant myeloperoxidase used for the first time in this kind of study was as convenient for pharmacological purposes as the purified one.

Bovine lactoperoxidase and its recombinant: comparison of structure and some biochemical properties.

Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.

The endothelium and cytokine secretion: the role of peroxidases as immunoregulators.

The endothelium is frequently exposed to many proinflammatory mediators. The present study was done to determine the effects of human recombinant myeloperoxidase (MPO) and porcine eosinophil peroxidase (EPO) on certain endothelial cell (HUVEC) functions. The following areas were evaluated: (1) production of reactive oxygen intermediates (ROI), (2) cytokine secretion, and (3) regulation of mRNA cytokine transcripts. Both MPO and EPO induced the production of ROI, but an enzymatically inactive form of MPO (iMPO) was the most effective. Enzymatically inactive MPO, but not MPO, induced the secretion of interleukins 6 and 8 and granulocyte-monocyte colony-stimulating factor. A ribonuclease protection assay indicated that both iMPO and MPO upregulated mRNA cytokine transcripts; however, the former was markedly more effective. The simultaneous addition of EPO and iMPO resulted in a decrease in cytokine-specific mRNA. These data indicate a major role for peroxidases in the regulation of inflammation.

A human milk factor susceptible to cathepsin D inhibitors enhances human immunodeficiency virus type 1 infectivity and allows virus entry into a mammary epithelial cell line.

Human immunodeficiency virus type 1 (HIV-1) growth in lymphocyte cultures was increased when the virus inoculum was incubated in breast milk. The enhancing effect of milk was abolished by anti-cathepsin D antibody or by pepstatin A, a cathepsin D inhibitor. The cathepsin D-producing CD4-negative MCF7 mammary cells supported the growth of some HIV-1 isolates. An MCF7 line chronically producing HIV-1 IIIb was obtained. Cathepsin D may induce conformational modification of viral gp120, allowing direct interaction with a coreceptor. We demonstrated the presence of CXCR4 mRNA in MCF7 cells.

Role of charged amino acids conserved in the vasoactive intestinal polypeptide/secretin family of receptors on the secretin receptor functionality.

The secretin receptor is a member of a large family of G-protein-coupled receptors that recognize polypeptide hormone and/or neuropeptides. Charged, conserved residues might play a key role in their function, either by interacting with the ligand or by stabilizing the receptor structure. Of the four charged amino acids that are conserved in the whole secretin receptor family, D49 and R83 (in the N-terminal domain) were probably important for the secretin receptor structure: replacement of D49 by H or R and of R83 by D severely reduced both the maximal response to secretin and its potency. No functional secretin receptor could be detected after replacement of R83 by L. Mutation of D49 to E, A, or N had no effect or reduced 5-fold the potency of secretin. The highly conserved positive charges found at the extracellular ends of TM III (K194) and IV (R255) were important for the secretin receptor function, as K194 mutation to A or Q and R255 mutation to Q or D decreased the secretin's affinity 15- to 1000-fold, respectively. Six extracellular charged residues are conserved in closely related receptors but not in the whole family. K121 (TM I) and R277 (TM V) were not important for functional secretin receptor expression. D174 (TM II) was necessary to stabilize the active receptor structure: the D174N mutant receptors were unable to stimulate normally the adenylate cyclase in response to secretin, and functional D174A receptors could not be found. Mutation of R255, E259 (second extracellular loop), and E351 (third extracellular loop) to uncharged residues reduced only 10- to 100-fold the secretin potency without changing its efficacy: these residues either stabilized the active receptor conformation or formed hydrogen rather than ionic bonds with secretin. Mutation of K121 (TM I) to Q or L and of R277 (TM V) to E or Q did not affect the receptor functional properties.

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.

Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.

The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found.

Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor.

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

Properties of a recombinant human secretin receptor: a comparison with the rat and rabbit receptors.

A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg16]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors.