Neutrophil extracellular traps promote cancer-associated inflammation and myocardial stress.

Cancer is associated with systemic pathologies that contribute to mortality, such as thrombosis and distant organ failure. The aim of this study was to investigate the potential role of neutrophil extracellular traps (NETs) in myocardial inflammation and tissue damage in treatment-naïve individuals with cancer. Mice with mammary carcinoma (MMTV-PyMT) had increased plasma levels of NETs measured as H3Cit-DNA complexes, paralleled with elevated coagulation, compared to healthy littermates. MMTV-PyMT mice displayed upregulation of pro-inflammatory markers in the heart, myocardial hypertrophy and elevated cardiac disease biomarkers in the blood, but not echocardiographic heart failure. Moreover, increased endothelial proliferation was observed in hearts from tumor-bearing mice. Removal of NETs by DNase I treatment suppressed the myocardial inflammation, expression of cardiac disease biomarkers and endothelial proliferation. Compared to a healthy control group, treatment-naïve cancer patients with different malignant disorders had increased NET formation, which correlated to plasma levels of the inflammatory marker CRP and the cardiac disease biomarkers NT-proBNP and sTNFR1, in agreement with the mouse data. Altogether, our data indicate that NETs contribute to inflammation and myocardial stress during malignancy. These findings suggest NETs as potential therapeutic targets to prevent cardiac inflammation and dysfunction in cancer patients.

Local delivery of temozolomide via a biologically inert carrier (Temodex) prolongs survival in glioma patients, irrespectively of the methylation status of MGMT.

Glioma is the most common brain malignancy. Standard first-line therapy for glioma includes surgery, radiotherapy and systemic administration of temozolomide. However, temozolomide does not reach the brain in sufficient doses when administered orally and has poor efficiency in more than half of the patients. Strategies to improve the treatment of glial malignancies are therefore needed. We have recently developed a system (Temodex) for local administration of temozolomide by encapsulating the drug in a biologically inert matrix. Here, we assessed the effect of Temodex in combination with standard therapy in a small-scale clinical study. Since the efficacy of temozolomide therapy is known to depend on the methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) promoter, we also analyzed whether the effect of Temodex was influenced by the methylation status of MGMT. Our data show that the combination of standard therapy and Temodex was more efficient than standard therapy alone, promoting the overall patient survival by up to 33 weeks. Moreover, the efficacy of Temodex was not dependent on the methylation status of MGMT. Local Temodex administration in combination with standard therapy thereby emerges as a novel therapeutic option, with applicability that is independent on the methylation status of the MGMT promoter.

Siramesine causes preferential apoptosis of mast cells in skin biopsies from psoriatic lesions.

BACKGROUND: Skin mast cells are implicated as detrimental effector cells in various inflammatory skin diseases such as contact eczema, atopic dermatitis and psoriasis. Selective reduction of cutaneous mast cells, e.g. by inducing targeted apoptosis, might prove a rational and efficient therapeutic strategy in dermatoses negatively influenced by mast cells. OBJECTIVES: The objective of the present study was to evaluate whether a lysosomotropic agent such as siramesine can cause apoptosis of mast cells present in psoriatic lesions. MATERIALS AND METHODS: Punch biopsies were obtained from lesional and uninvolved skin in 25 patients with chronic plaque psoriasis. After incubation with siramesine, the number of tryptase-positive mast cells and their expression of interleukin (IL)-6 and IL-17 was analysed. Skin biopsies were digested to allow flow cytometric analysis of the drug's effect on cutaneous fibroblasts and keratinocytes. RESULTS: Siramesine caused a profound reduction in the total number of mast cells in both lesional and uninvolved psoriatic skin biopsies without affecting the gross morphology of the tissue. The drug reduced the density of IL-6- and IL-17-positive mast cells, and showed antiproliferative effects on epidermal keratinocytes but had no apparent cytotoxic effect on keratinocytes or dermal fibroblasts. CONCLUSIONS: Considering the pathophysiology of psoriasis, the effects of siramesine on cutaneous mast cells may prove favourable from the therapeutic aspect. The results encourage further studies to assess the usefulness of siramesine and other lysosomotropic agents in the treatment of cutaneous mastocytoses and inflammatory skin diseases aggravated by dermal mast cells.

Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine in vivo-derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15-100 µm for up to 48 h. The overall cell viability was unaffected by 15 µm LLME up to 48 h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24 h of incubation with 100 µm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.

Mast cell chymase modulates IL-33 levels and controls allergic sensitization in dust-mite induced airway inflammation.

Mast cells (MCs) are major effector cells contributing to allergic conditions. When activated, they can release large amounts of active proteases, including chymase from their secretory granules. Here we assessed the role of the chymase mouse mast cell protease 4 (mMCP-4) in allergic airway inflammation induced by house-dust mite (HDM) extract. mMCP-4-/-) mice demonstrated elevated airway reactivity and eosinophilia compared with wild-type (WT) animals, suggesting a protective role for mMCP-4 during the late inflammatory phase of the disease. However, mMCP-4 also contributed to the sensitization phase, as indicated by higher levels of serum immunoglobulin E in mMCP-4(-/-) vs. WT mice and higher levels of cytokines secreted by HDM-restimulated mMCP-4(-/-) vs. WT splenocytes. In line with a contribution of mMCP-4 in the early stages of disease, HDM extract directly induced chymase secretion from MCs. The elevated airway and inflammatory responses of mMCP-4(-/-) mice were associated with a profound increase in the levels of interleukin (IL)-33 in the lung tissue. Moreover, WT MCs degraded IL-33 more efficiently than did MCs lacking mMCP-4. Together, our findings identify a protective role of a MC chymase in a physiologically relevant model for airway inflammation and suggest that chymase-mediated regulation of IL-33 can account for this protective function.

Lysosomal membrane permeabilization induces cell death in human mast cells.

Mast cells (MC) have pathogenic roles in numerous disorders, and strategies that stabilize MC or induce MC apoptosis are therefore emerging as possible therapeutic regimens. A typical feature of MC is their high content of secretory lysosomes (granules), containing numerous components such as biogenic amines, cytokines, serglycin proteoglycan and proteases. Damage to the secretory lysosomes will thus lead to leakage of these compounds, including the proteases, into the cytosol, and this could potentially trigger apoptosis. Here, we evaluated whether MC are sensitive to cell death induced by secretory lysosome destabilization, induced by the lysosomotropic agent Leu-Leu-OMe (LLME). Human MC were sensitive to LLME-induced cell death. In contrast, fibroblasts and HEK-293 cells were largely resistant. As judged by Annexin V/propidium iodide staining, LLME caused apoptotic cell death, and this was supported by induction of caspase-3-like activity, detection of activated caspase-3 by immunoblot analysis and reduced cell death in the presence of a caspase inhibitor. In support of a role for serglycin in regulating LLME-induced cell death, the survival rate of various cell types correlated negatively with the level of serglycin expression. In summary, this study introduces the concept of using lysosomotropic agents to induce cell death of human MC.

Plasticity of cloned canine mammary spindle cell tumor, osteosarcoma and carcinoma cells.

Female dogs are frequently affected by mammary tumors, both carcinomas and sarcomas. The mechanisms behind mammary-tumor formation and the high degree of heterogeneity are not understood. To provide insight into this issue, it is important to determine the properties of the cells forming the different types of tumors. One question is if individual neoplastic cells can give rise to phenotypically distinct tumor types, i.e., show plasticity. We studied 3 different tumors (a spindle-cell tumor, an osteosarcoma, and a carcinoma) and followed the change of lineage marker expression between the primary canine mammary tumors, the clones derived from the corresponding tumors and in tumors generated after inoculation of tumor clones into nude mice (n = 75). Inoculation of clones derived from the spindle-cell tumor gave rise to spindle-cell tumors in nude mice. Several of these contained bone tissue, a sign of plasticity. Clones derived from the osteosarcoma were negative for a panel of lineage markers but, when inoculated into nude mice, they were able to form bone, again a sign of plasticity. In contrast to the primary carcinoma, most of the clones derived thereof lacked keratin expression, but keratin expression was recovered in most of the tumors formed after inoculation of clones into nude mice. Moreover, tumors generated from the carcinoma clones, in contrast to the primary tumor, were positive for smooth-muscle-cell markers. Our results point to plasticity in canine mammary tumors, as shown both by morphologic criteria and by expression patterns for lineage specific markers.

Regulation of extravascular coagulation and fibrinolysis by heparin-dependent mast cell chymase.

We recently characterized a heparin-deficient mouse strain generated by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2-/- mice show severe defects in their organization of mast cell (MC) secretory granules, with an almost total absence of the various heparin-binding MC proteases. In the present report we have studied the consequences of heparin/MC protease deficiency for extravascular coagulation and fibrinolysis. Addition of prothrombin to peritoneal cells-a mixture of macrophages, lymphocytes, and MCs-resulted in formation of thrombin but the accumulation of thrombin occurred faster in the NDST-2-/-cells than in normal controls. Further, the generated thrombin was subsequently inactivated in the NDST-2+/+ cell cultures but not in the NDST-2-/- cells. Plasminogen was activated to plasmin at an apparently higher rate in peritoneal cells from NDST-2 null mice than in the normal controls. Similar to thrombin, the generated plasmin was inactivated by NDST-2+/+ but not by the NDST-2-/- cells. Subsequent experiments with normal cells showed that cell surface-associated MC chymase, in a strongly heparin-dependent manner, was responsible for both the thrombin-inactivating- and plasmin-inactivating activities. These results show that MC chymase-heparin complexes have the potential to regulate extravascular coagulation processes, as well as the plasminogen activator/plasmin system.

Structural requirements and mechanism for heparin-induced activation of a recombinant mouse mast cell tryptase, mouse mast cell protease-6: formation of active tryptase monomers in the presence of low molecular weight heparin.

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase. Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active tryptase monomers that may be formed according to the mechanism described here.

Mice lacking histidine decarboxylase exhibit abnormal mast cells.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Human peripheral blood mononuclear cells require factor X and platelets for expression of prothrombinase activity in response to bacterial lipopolysaccharide.

We investigated whether human peripheral blood mononuclear cells (PBMC) express prothrombinase following stimulation with bacterial lipopolysaccharide (LPS). LPS-stimulated PBMC devoid of contaminating platelets failed to activate prothrombin directly. Addition of platelets did not result in expression of prothrombinase in the absence of factor X whereas the combination of platelets and factor X resulted in strong prothrombinase activity on LPS-activated cells. The induced prothrombinase was dependent on tissue factor, as the activity was completely inhibited by an anti-tissue factor antibody. Our data suggest that platelet/monocyte cooperation is important in the generation of prothrombinase activity in response to endotoxin.

Heparin antagonists are potent inhibitors of mast cell tryptase.

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.

Altered processing of fibronectin in mice lacking heparin. a role for heparin-dependent mast cell chymase in fibronectin degradation.

We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.

Macrophages synthesize factor X and secrete factor X/Xa-containing prothrombinase activity into the surrounding medium.

Activation of the coagulation cascade, mediated by various monocyte/macrophage procoagulants, is an important component in the pathology of inflammatory disease. The type of procoagulant expressed may vary between different monocyte/macrophage subtypes and may differ depending on how the cells are treated. In the present study we show that both murine peritoneal macrophages and human adherent synovial cells from rheumatoid arthritis lesions express prothrombinase activity that was inhibited by anti-Factor X antibodies. Northern blot analysis showed that Factor X was transcribed by the murine peritoneal cells and Western blot analysis showed the presence of Factor X antigen. Further experiments showed that the prothrombinase activity was secreted by the cells into the medium in a detergent-sensitive form, suggesting that the prothrombinase is released on small lipid-containing vesicles.

Mechanism by which heparin proteoglycan modulates mast cell chymase activity.

Chymases are highly basic chymotrypsin-like serine proteases expressed exclusively by mast cells. Large amounts of chymases complexed with heparin proteoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the protease toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates. Incubation of thrombin with oligonucleotides that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants with defects in their heparin-binding regions were less efficiently inactivated by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-catalyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptide substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, whereas the cleavage of uncharged substrates was not affected by the presence of heparin PG. On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrates.

Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme.

Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions. The initial modification step, catalysed by the enzyme glucosaminyl N-deacetylase/N-sulphotransferase-2, NDST-2, is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.

Procoagulant and anticoagulant activities of resident and inflammatory peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the involvement of both procoagulants and anticoagulants from adherent peritoneal cells in an inflammatory model was investigated. MATERIAL AND TREATMENT: Mice were injected with thioglycollate broth or lipopolysaccharide (LPS). METHODS: Cells were harvested by peritoneal lavage. Adherent peritoneal cells were cultured further +/- LPS. Subsequently, their ability to activate either Factor X or prothrombin, and their ability to inactivate thrombin through a thrombin-degrading mast cell chymase, was assayed. RESULTS: Inflammatory cells expressed reduced amounts of thrombin-inactivating activity as compared with control resident peritoneal cells. Both resident and inflammatory cells expressed potent prothrombinase activities. Further stimulation of the various cellular populations with LPS in vitro had very little effect on the prothrombinase and thrombin-inactivating activities, bud had a strong stimulatory effect on the Factor X-activating activities.

Vitronectin expression in rheumatoid arthritic synovia--inhibition of plasmin generation by vitronectin produced in vitro.

The plasmin-generating system controls, to a great extent, the degree of connective tissue destruction as well as fibrin deposition two contributors to the pathogenesis generated in diseases such as rheumatoid arthritis. Vitronectin, an adhesive blood glycoprotein, has the potential to modulate this system by its known capacity to interact with plasminogen activator inhibitor-1, plasminogen activators, the urokinase plasminogen activator receptor, and plasminogen. The net effect of these interactions, in terms of plasmin generation, is not known as yet. In the present study, we have investigated the possible expression and role of vitronectin in rheumatoid arthritic synovia. Analysis of synovial frozen sections by immunofluorescence showed the presence of vitronectin in the 13 cases studied. In situ hybridization analysis demonstrated the presence of vitronectin mRNA in cells present in areas rich in infiltrating inflammatory cells. The adherent population of the rheumatoid arthritic synovial cells was isolated and found to synthesize and secrete vitronectin into the medium (seven of 10 isolates), as assessed by metabolic labelling and immunoprecipitation. Plasmin-generating activity was detected in the adherent synovial cells, and this activity was increased by antibodies to vitronectin. Our findings show, for the first time, that vitronectin can be endogenously produced in a pathophysiological system where it can inhibit the generation of plasmin.

Cloning, structural analysis, and expression of the pig IgE epsilon chain.

As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the epsilon chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the epsilon chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian epsilon-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE epsilon chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 x His-CH2-CH3-CH4 protein product.