Ceruloplasmin and myeloperoxidase in complex affect the enzymatic properties of each other

Ceruloplasmin (CP), the multicopper oxidase of plasma, interacts with myeloperoxidase (MPO), an enzyme of leukocytes, and inhibits its peroxidase and chlorinating activity. Studies on the enzymatic properties shows that CP behaves as a competitive inhibitor impeding the binding of aromatic substrates to the active centre of MPO. The contact between CP and MPO probably entails conformational changes close to the p-phenylenediamine binding site in CP, which explains the observed activation by MPO of the substrate's oxidation. CP subjected to partial proteolysis was virtually unable to inhibit activity of MPO. The possible protein–protein interface is comprised of the area near active site of MPO and the loop linking domains 5 and 6 in CP. One of the outcomes of this study is the finding of a new link between antioxidant properties of CP and its susceptibility to proteolysis.     

Identification and properties of complexes formed by myeloperoxidase with lipoproteins and ceruloplasmin

The first evidence of multi-component complexes formed by myeloperoxidase (MPO), ceruloplasmin (CP), and very low/low density lipoproteins (VLDL/LDL) obtained by electrophoresis, gel filtration, and photon-correlation spectroscopy (PCS) is presented in this paper. Complexes were observed when isolated MPO, CP, and VLDL/LDL were mixed and/or when MPO was added to the blood plasma. Complex LDL–MPO–CP was detected in 44 of 100 plasma samples taken from patients with atherosclerosis, and 33 of 44 samples also contained the VLDL–MPO–CP complex. MPO concentration in these patients’ plasma exceeded 800 ng/ml. Interaction of MPO with high density lipoproteins (HDL) was not revealed, as well as binding of CP to lipoproteins in the absence of MPO. Adding antibodies against apoB-100 to VLDL–MPO–CP and LDL–MPO–CP complexes results in release of lipoproteins. Using PCS the diameters of complexes under study were evaluated. By comparing concentrations of the components in complexes formed by MPO, CP, and lipoproteins their stoichiometry was assessed as 2VLDL:1MPO:2CP and 1LDL:1MPO:2CP. Lipoproteins affected the inhibition of MPO peroxidase activity by CP. The affinity of lipoproteins to MPO–CP complex was assessed using apparent dissociation constants determined as ～0.3 nM for VLDL and ～0.14 nM for LDL.     

pH-dependent regulation of myeloperoxidase activity

The balance between peroxidase and chlorinating activities of myeloperoxidase (MPO) is very important for the enhancement of antimicrobial action and prevention of damage caused by hypochlorite. In the present paper, the peroxidase and chlorinating activities have been studied at various pH values. The possibility of using neutrophil protein solution for the evaluation of MPO activity has been demonstrated. It is shown that at neutral pH MPO had higher affinity to peroxidase substrate guaiacol: at pH 7.4, chloride ions did not compete with guaiacol up to the concentration of 150 mM. At acidic pH, chlorinating activity of MPO dominates: only hypochlorite production can be detected at equal chloride and guaiacol concentrations of 15 mM. However, horseradish peroxidase does not exhibit any difference in activity in the presence of chloride ions even at acidic pH values. It was demonstrated by MALDI-TOF mass-spectrometry that the amount of hypochlorite produced is sufficient to modify phospholipids (with formation of Cl- and Br-hydrins and lyso-derivatives) only at acidic pH (5.0). Thus, in the presence of phenolic peroxidase substrate, MPO chlorinating activity can be displayed at acidic pH only. It can lead to elimination of hypochlorite production in normal tissues at neutral pH (7.4) and its enhancement in phagosomes where the pH range is 4.7-6.0.     

Thrombin inhibits the anti-myeloperoxidase and ferroxidase functions of ceruloplasmin: relevance in rheumatoid arthritis

Human ceruloplasmin (CP) is a multifunctional copper-binding protein produced in the liver. CP oxidizes Fe²⁺ to Fe³⁺, decreasing the concentration of Fe²⁺ available for generating harmful oxidant species. CP is also a potent inhibitor of leukocyte myeloperoxidase (MPO) (K_d=130 nM), a major source of oxidants in vivo. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting flexible joints and characterized by activation of both inflammatory and coagulation processes. Indeed, the levels of CP, MPO, and thrombin are markedly increased in the synovial fluid of RA patients. Here we show that thrombin cleaves CP in vitro at ⁴⁸¹Arg–Ser⁴⁸² and ⁸⁸⁷Lys–Val⁸⁸⁸ bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the MPO inhibitory function of CP is abrogated. Analysis of the synovial fluid of 24 RA patients reveals that CP is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with thrombin in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. Using independent biophysical techniques, we show that thrombin has intrinsic affinity for CP (K_d=60–270 nM), independent of proteolysis, and inhibits CP ferroxidase activity (K_I=220±20 nM). Mapping of thrombin binding sites with specific exosite-directed ligands (i.e., hirugen, fibrinogen γ′-peptide) and thrombin analogues having the exosites variably compromised (i.e., prothrombin, prethrombin-2, β_T-thrombin) reveals that the positively charged exosite-II of thrombin binds to the negatively charged upper region of CP, while the protease active site and exosite-I remain accessible. These results suggest that thrombin can exacerbate inflammation in RA by impairing the MPO inhibitory function of CP via proteolysis and by competitively inhibiting CP ferroxidase activity.Notably, local administration of hirudin, a highly potent and specifc thrombin inhibitor, reduces the concentration of active MPO in the synovial fluid of RA patients and has a beneficial effect on the clinical symptoms of the disease.     

Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes

We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H₂O₂), largely reduced the active force (F_active), increased the passive force (F_passive), and decreased the Ca²⁺ sensitivity of force production (pCa₅₀) in permeabilized cardiomyocytes. H₂O₂ alone had significantly smaller effects on F_active and F_passive and did not alter pCa₅₀. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H₂O₂ alone and MPO + H₂O₂ reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca²⁺-regulated active and Ca²⁺-independent passive force production and myofilament Ca²⁺ sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.     

Influence of ceruloplasmin and lactoferrin on the chlorination activity of leukocyte myeloperoxidase assayed by chemiluminescence

We demonstrate that addition of H₂O₂ to a mixture of myeloperoxidase (MPO), chloride and luminol immediately evokes a short intense flash of chemiluminescence (CL). This flash is diminished in the absence of MPO or chloride, and in the complete system it is suppressed by an MPO inhibitor azide, hypochlorite scavengers taurine or methionine, or an MPO peroxidase-cycle substrate guaiacol. Hence, this CL is mostly due to the MPO halogenation function; a measure of this activity is provided by the integral CL. With three independent methods (CL, taurine chlorination, and peroxidase assay) it is shown that MPO activity is suppressed by ceruloplasmin (Cp). Lactoferrin has no effect either on MPO or on the MPO-Cp complex. It is also shown that peroxidase inhibition by Cp is the stronger the larger is the MPO substrate, which suggests steric hindrances to substrate binding in the MPO-Cp complex. Importantly, the conventional chlorination and peroxidase assays detect MPO inhibition by Cp only at a large excess of the latter, whereas the CL assay reveals it at stoichiometric ratios characteristic of the naturally occurring protein complexes.     

Study of Interaction of Ceruloplasmin with Serprocidins

This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1: 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K _i ≈ 1 μM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K _i ≈20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5–13, 61–63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.     

Enzymatic and Bactericidal Activity of Monomeric and Dimeric Forms of Myeloperoxidase

Enzymatic and bactericidal activities of mature, dimeric myeloperoxidase (MPO) and its monomeric form have been compared. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each by two methods. Bactericidal activity of the MPO/Н₂О₂/Cl￣ system was tested using the Escherichia coli laboratory strain DH5α. No difference in the enzymatic and bactericidal activity between dimeric MPO and hemi- MPO was found. Both forms of the enzyme also did not differ in the resistance to HOCl, the main product of MPO. HOCl caused a dose-dependent decrease in peroxidase and chlorinating activity, and the pattern of this decrease was identical for dimeric MPO and hemi-MPO. At the equal heme concentration, the hemi- MPO/Н₂О₂/Cl￣ system demonstrated a somewhat higher bactericidal effect than the dimeric MPO/Н₂О₂/Cl￣ system. This is most likely explained by higher probability of contacts between the bacterial surface and hemi-MPO molecules, since at the same heme concentration the number of hemi-MPO molecules is 2-fold higher than that of dimeric MPO molecules. Using Western-blotting with antibodies to MPO, we have shown, for the first time, that the dimeric molecule of MPO could be cleaved into two monomeric subunits by HOCl, most probably due to oxidation of the disulfide bond between these subunits. This suggests that appearance in blood of MPO with mass corresponding to its monomer may result from the damage of dimeric MPO by reactive halogen species, especially upon their overproduction inducing oxidative/halogenative stress in inflammatory diseases.     

The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase

A key function of neutrophil myeloperoxidase (MPO) is the synthesis of hypochlorous acid (HOCl), a potent oxidizing agent that plays a cytotoxic role against invading bacteria and viruses at inflammatory sites and in phagosomes. MPO displayed a chlorinating activity preferably at acidic pH but at neutral pH MPO catalyzes mainly reactions of the peroxidase cycle. In the present work effects of tyrosine on the chlorinating activity of MPO were studied. At pH 7.4 we detected an increased HOCl production in the presence of tyrosine not only by the MPO-H₂O₂-Cl^- system but also in suspensions of zymosan-activated neutrophils. An excess of H₂O₂ is known to cause an accumulation of compound II of MPO blocking the generation of HOCl at neutral pH. As evidenced by spectral changes, tyrosine-induced activation of MPO to synthesize HOCl was due to the ability of tyrosine to reduce compound II back to the native state, thus accelerating the enzyme turnover. MPO-induced oxidation of tyrosine is relevant to what can be in vivo; we detected MPO-catalyzed formation of dityrosine in the presence of plasma under experimental conditions when tyrosine concentration was about three magnitudes of order less than the Cl⁻ concentration. At acidic pH formation of compound II was impaired in the presence of chloride and dityrosine couldn't be detected in plasma. In conclusion, the ability of tyrosine to increase the chlorinating activity of MPO at neutral pH and enhanced values of H₂O₂ may be very effective for the specific enhancement of HOCl production under acute inflammation.     

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.     

Myeloperoxidase-catalyzed iodination and coupling.

Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.     

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase,built on the scaffold of the myeloperoxidase X-ray structure

 Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed. Received: 2 May 1996 / Accepted: 25 July 1996     

The role of CP 47 in the evolution of oxygen and the binding of the extrinsic 33-kDa protein to the core complex of Photosystem II as determined by limited proteolysis

In order to identify the domain within Photosystem II complexes that functions in the evolution of oxygen, we performed limited proteolysis with lysylendopeptidase of the core complex of Photosystem II which had been depleted of the extrinsic 33-kDa protein (Mn-stabilizing protein). The cleavage sites were estimated from the amino-terminal sequences of the degradation fragments, their apparent molecular masses and amino-acid compositions. Under certain conditions, the D2 protein was cleaved at Lys13; and a chlorophyll a-binding protein, CP 47, was cleaved at Lys227 and Lys389. Another chlorophyll a-binding protein, CP 43, was degraded more rapidly than CP 47. The oxygen-evolving activity and the capacity for rebinding of the 33-kDa protein to the core complex of Photosystem II decreased in parallel, with kinetics very similar to those of the cleavage of CP 47 at Lys389. These observations strongly suggest that the hydrophilic domain around Lys389 of CP 47, which are located on the lumenal side, is important in the binding of the 33-kDa protein and in maintaining the oxygen-evolving activity of the Photosystem II complex.Abbreviations CP 47 and CP 43- intrinsic chlorophyll a-binding proteins with apparent molecular masses of 47 and 43 kDa, respectively - PBQ- phenyl-p-benzoquinone - TLCK- N--p-tosyl-L-lysine chloromethyl ketone     

Study of interaction of ceruloplasmin,lactoferrin, and myeloperoxidase by photon correlation spectroscopy

In this work, the diameters of protein complexes formed upon interaction of ceruloplasmin (CP) with lactoferrin (LF) and myeloperoxidase (MPO) were determined. Gage dependence of the diameter of protein particles (myoglobin, albumin, LF, CP, MPO, aldolase, ferritin) on their molecular mass logarithm was calculated. The diameter of a complex formed upon mixing CP and LF was 8.4 nm, which is in line with the radius of gyration obtained previously when the 1CP-1LF complex was studied by small-angle X-ray scattering. The diameter of a complex formed upon interaction of CP with MPO is 9.8 nm, corresponding to the stoichiometry 2CP: 1MPO. The diameter of a complex formed when LF is added to the 2CP-1MPO complex is 10.7 nm. The latter is consistent with the notion of a pentameric structure 2LF-2CP-1MPO with molecular mass of about 585 kDa.     

Interaction of ceruloplasmin, lactoferrin, and myeloperoxidase

When lactoferrin (LF) and myeloperoxidase (MPO) are added to ceruloplasmin (CP), a CP-LF-MPO triple complex forms. The complex is formed under physiological conditions, but also in the course of SDS-free PAGE. Polyclonal antibodies to both LF and MPO displace the respective proteins from the CP-LF-MPO complex. Similar replacement is performed by a PACAP38 fragment (amino acids 29-38) and protamine that bind to CP. Interaction of LF and MPO with CP-Sepharose is blocked at ionic strength above 0.3 M NaCl and at pH below 4.1 (LF) and 3.9 (MPO). Two peptides (amino acids 50-109 and 929-1012) were isolated by affinity chromatography from a preparation of CP after its spontaneous proteolytic cleavage. These peptides are able to displace CP from its complexes with LF and MPO. Both human and canine MPO could form a complex when mixed with CP from seven mammalian species. Upon intravenous injection of human MPO into rats, the rat CP-human MPO complex could be detected in plasma. Patients with inflammation were examined and CP-LF, CP-MPO, and CP-LF-MPO complexes were revealed in 80 samples of blood serum and in nine exudates from purulent foci. These complexes were also found in 45 samples of serum and pleural fluid obtained from patients with pleurisies of various etiology.     

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix

Background

Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood.

Methods

We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested.

Results

MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed.

Conclusions

Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins.

General significance

Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.     

Thiocyanate modulates the catalytic activity of mammalian peroxidases

We investigated the potential role of the co-substrate, thiocyanate (SCN-), in modulating the catalytic activity of myeloperoxidase (MPO) and other members of the mammalian peroxidase superfamily (lactoperoxidase (LPO) and eosinophil peroxidase (EPO)). Pre-incubation of SCN- with MPO generates a more complex biological setting, because SCN- serves as either a substrate or inhibitor, causing diverse impacts on the MPO heme iron microenvironment. Consistent with this hypothesis, the relationship between the association rate constant of nitric oxide binding to MPO-Fe(III) as a function of SCN- concentration is bell-shaped, with a trough comparable with normal SCN- plasma levels. Rapid kinetic measurements indicate that MPO, EPO, and LPO Compound I formation occur at rates slower than complex decay, and its formation serves to simultaneously catalyze SCN- via 1e- and 2e- oxidation pathways. For the three enzymes, Compound II formation is a fundamental feature of catalysis and allows the enzymes to operate at a fraction of their possible maximum activities. MPO and EPO Compound II is relatively stable and decays gradually within minutes to ground state upon H2O2 exhaustion. In contrast, LPO Compound II is unstable and decays within seconds to ground state, suggesting that SCN- may serve as a substrate for Compound II. Compound II formation can be partially or completely prevented by increasing SCN- concentration, depending on the experimental conditions. Collectively, these results illustrate for the first time the potential mechanistic differences of these three enzymes. A modified kinetic model, which incorporates our current findings with the mammalian peroxidases classic cycle, is presented.     

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K _i for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A ₆₁₀/A ₂₈₀ ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.     

Uptake of human eosinophil peroxidase and myeloperoxidase by cells involved in the inflammatory process

We have recently shown that human neutrophils bind and internalize human eosinophil peroxidase (EPO) but not myeloperoxidase (MPO). In the present work, we studied the interactions of human EPO and MPO with other cells that may be involved in the inflammatory process, i.e., lymphocytes, monocytes, platelets, fibroblasts, and endothelial cells. The results indicate that EPO is bound by all the cell types considered, but is efficiently internalized only by lymphocytes, monocytes, and endothelial cells. Conversely, MPO binds appreciably only to fibroblasts and endothelial cells, although with a lower affinity than EPO, but its internalization by any of the cell types studied is hardly detectable. Furthermore, both peroxidases bind strongly to collagen fibers, whereas only EPO binds to elastin. The results suggest that EPO, owing to its high cytophilia, exerts its biological activity close to the site at which it is released from the eosinophil.