Denise Schlorke

The influence of glycosaminoglycans on IL-8-mediated functions of neutrophils.

Glycosaminoglycans (GAGs) of the extracellular matrix (ECM) contribute to the regulation of physiological processes by binding various immune-competent proteins. Due to their large structural diversity, the analysis of the binding properties and their functional consequences is challenging. The cytokine interleukin-8 (IL-8) is involved in the recruitment of neutrophils to inflammatory sites. Here, we investigated the interaction of heparin hexasaccharides and recombinant human IL-8, consisting of 77 amino acids using fluorescence and NMR spectroscopy. A dissociation constant of 2.0±0.4 μM was determined for the heparin-IL-8 complex, which is slightly higher than what has been found for chondroitin-6-sulfate (K(D)=1.4±0.4 μM) [Pichert, A.; Samsonov, S. A.; Theisgen, S.; Thomas, L.; Baumann, L.; Schiller, J.; Beck-Sickinger, A. G.; Huster, D.; Pisabarro, M. T. Glycobiology2012, 22, 134-145], suggesting an important role of the sulfate group at position 6 of the second ring in the disaccharide unit of the GAGs in this interaction. In addition, the influence of long-chain hyaluronan, chondroitin sulfate, and heparin on IL-8-induced chemotaxis and oxidative activity of neutrophils was examined. Only the incubation of heparin with IL-8 affected the IL-8-mediated chemotaxis of neutrophils. However, all investigated GAGs enhanced the IL-8-induced formation of reactive oxygen species in neutrophils, which is an entirely new finding. This work provides a representative example of how protein functions can be regulated by different GAGs of the ECM.

Functional aspects of the interaction between interleukin-8 and sulfated glycosaminoglycans.

During the immune response, the cytokine interleukin 8 (IL-8, CXCL8) functions as a strong chemoattractant for polymorphonuclear leukocytes helping to direct these cells to infected/injured sites. This review focuses on the interaction of IL-8 with sulfated glycosaminoglycans expressed on cell surfaces and the extracellular matrix. This interaction contributes to the recruitment of polymorphonuclear cells from blood, penetration of these cells through the vessel wall, and their directed migration to inflammatory sites. Regulatory aspects of the interplay between IL-8 and heparan sulfate, the most abundant glycosaminoglycan, are highlighted. In this field, the large natural heterogeneity of glycosaminoglycans represents a great challenge that impedes the modeling of IL-8 functions. The interaction of IL-8 with newly developed artificial sulfated hyaluronan derivatives is also considered as these artificial substrates are an important tool for development of new materials in regenerative medicine.

Lactoperoxidase as a potential drug target

Introduction: Lactoperoxidase (LPO) belongs to the immunologically relevant mammalian heme peroxidases. The enzyme contributes in external secretions to the humoral immune defense against pathogens by oxidation of thiocyanate (SCN–) and iodide (I–). The generation of oxidized thiocyanate and/or iodine species is also important in numerous biotechnological applications of LPO. Areas covered: In this review, we give an overview about the present knowledge of LPO concerning enzymatic structure, catalytic cycles and (pseudo-)halogenated species generated by the enzyme. Redox properties of LPO as well as kinetic aspects regarding the different enzymatic cycles are discussed in order to gain insights into the disturbance of the (pseudo-)halogenating enzyme activity under pathological conditions. Important structural features of LPO and crystallographic studies on the interaction and reaction of organic substrates with the enzyme are also summarized. A broad discussion is devoted to the binding and oxidation of substrates that either inhibit or promote LPO activity. Expert opinion: On the basis of these data, different strategies to further optimize LPO functions in humoral defense of mucous surfaces and biotechnological applications are discussed. In particular, hydrophobic organic substrates with a 3,4-dihydroxyphenyl partial structure considerably enhance the (pseudo-)halogenating activity of LPO. Their application provides, thus, a new strategy to enhance the anti-microbial activity of this enzyme.

Impact of cyanogen iodide in killing of Escherichia coli by the lactoperoxidase-hydrogen peroxide-(pseudo)halide system

Abstract In the presence of hydrogen peroxide, the heme protein lactoperoxidase is able to oxidize thiocyanate and iodide to hypothiocyanite, reactive iodine species, and the inter(pseudo)halogen cyanogen iodide. The killing efficiency of these oxidants and of the lactoperoxidase-H2O2-SCN−/I− system was investigated on the bioluminescent Escherichia coli K12 strain that allows time-resolved determination of cell viability. Among the tested oxidants, cyanogen iodide was most efficient in killing E. coli, followed by reactive iodine species and hypothiocyanite. Thereby, the killing activity of the LPO-H2O2-SCN−/I− system was greatly enhanced in comparison to the sole application of iodide when I− was applied in two- to twenty-fold excess over SCN−. Further evidence for the contribution of cyanogen iodide in killing of E. coli was obtained by applying methionine. This amino acid disturbed the killing of E. coli mediated by reactive iodine species (partial inhibition) and cyanogen iodide (total inhibition), but not by hypothiocyanite. Changes in luminescence of E. coli cells correlate with measurements of colony forming units after incubation of cells with the LPO-H2O2-SCN−/I− system or with cyanogen iodide. Taken together, these results are important for the future optimization of the use of lactoperoxidase in biotechnological applications.

Formation of cyanogen iodide by lactoperoxidase

The haem protein lactoperoxidase (LPO) is an important component of the anti-microbial immune defence in external secretions and is also applied as preservative in food, oral care and cosmetic products. Upon oxidation of SCN(-) and I(-) by the LPO-hydrogen peroxide system, oxidised species are formed with bacteriostatic and/or bactericidal activity. Here we describe the formation of the inter(pseudo)halogen cyanogen iodide (ICN) by LPO. This product is formed when both, thiocyanate and iodide, are present together in the reaction mixture. Using (13)C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry we could identify this inter(pseudo)halogen after applying iodide in slight excess over thiocyanate. The formation of ICN is based on the reaction of oxidised iodine species with thiocyanate. Further, we could demonstrate that ICN is also formed by the related haem enzyme myeloperoxidase and, in lower amounts, in the enzyme-free system. As I(-) is not competitive for SCN(-) under physiologically relevant conditions, the formation of ICN is not expected in secretions but may be relevant for LPO-containing products.

New insights into thiocyanate oxidation by human myeloperoxidase

Human myeloperoxidase (MPO) uses chloride and thiocyanate as physiological substrates at neutral pH. Oxidation of thiocyanate to hypothiocyanite mediated by the redox intermediate Compound I rapidly restores the ferric state of MPO. At low thiocyanate concentration and in the presence of hydrogen peroxide the observed reaction sequence is Compound I→ferric MPO→Compound II→MPO-cyanide complex, whereas at high thiocyanate concentrations and in the absence of H2O2 the only observed transition is Compound I→ferric MPO. The reaction of ferric MPO with hypothiocyanite directly forms the MPO-cyanide complex, whereas a transient product derived from the reaction between hypothiocyanite and hydrogen peroxide is demonstrated to mediate the conversion of ferric MPO to Compound II. Mechanisms for those reactions are discussed and proposed.

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Abstract Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.

Effects of WF10 on Glycosaminoglycan Sulphation in Proinflammatory Monocytes and Macrophages

The chlorite-based drug solution WF10 has been successfully applied to dampen strong inflammatory disease states and to improve wound healing processes. Howev‐ er, the molecular mechanisms of this drug are not well understood. This study is di‐ rected to investigate how WF10 and its components affect the expression of surface markers and sulphated proteoglycans and glycosaminoglycans in proinflammatorystimulated monocytes and macrophages. Human blood-derived macrophages were cultivated from monocytes in the presence of 50 U/ml granulocyte-macrophage colony-stimulating factor and activated by a mix‐ ture of 100 ng/ml lipopolysaccharide (LPS) and 10 ng/ml interferon γ (IFNγ). These cells were identified and characterised by their specific cell-surface receptors CD14, CD16, CD80, CD86, CD163, and CD206 using flow cytometry approaches. The sulpha‐ tion level of proteoglycans and glycosaminoglycans was assessed by the BlyscanTM dye-binding assay. The expression of the surface marker CD44, a proteoglycan with sulphated glycosaminoglycan side chains, was followed by antibodies against CD44. The binding of fluorescence-labelled hyaluronan to CD44 was also investigated by flow cytometry. All analyses were performed after incubation of monocytes and mac‐ rophages with WF10 or with its main components chlorite and chlorate. The drug substance WF10 inhibited the activation of LPS/IFNγ-stimulated human monocyte-derived macrophages. Among them are the diminished expression of proinflammatory surface markers, the inhibition of the expression of the hyaluronan receptor CD44, and the binding of hyaluronan to CD44. Further, the overall amount of sulphated proteoglycans and glycosaminoglycans was down-regulated by WF10. These in vitro experiments indicate that WF10 is able to inhibit the proinflammatory activation of macrophages. The results suggested that chlorite is the active principle in WF10 as chlorite caused principally the same changes in targets as WF10. The WF10 component chlorate inhibited only the overall sulphation level of proteoglycans and glycosaminoglycans and the binding of hyaluronan to CD44.

Early Events in Apoptosis Induction in Polymorphonuclear Leukocytes

Different white blood cells are involved in immune responses to pathogens, environmental stress, alterations in energy and nutrition supply as well as traumata. To find an adequate answer to the myriad of exogenous and endogenous noxes is a high challenge for the human immune system. Chronic inflammatory diseases like rheumatoid arthritis, arteriosclerosis, inflammatory bowel disease, and many others are associated with a disturbed regulation of immune functions (Peng, 2006; Zernecke & Weber, 2010). Despite numerous worldwide investigations regulatory aspects are only scarcely understood in innate and acquired immunity.