Effect of deuterium oxide on neutrophil oxidative metabolism,phagocytosis, and lysosomal enzyme release

We have previously shown that deuterium oxide (D₂O) enhances the oxidation of methionine, a myeloperoxidase (MPO)-mediated reaction, by human neutrophils during phagocytosis. However, D₂O has no effect on the oxidation of methionine by the purified MPO-H₂O₂-Cl^– system. To explain this observation, we studied the effect of D₂O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D₂O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D₂O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D₂O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H₂O₂ production by neutrophils in the presence of latex particles or PMA were also stimulated by D₂O. In contrast, D₂O inhibited the ingestion of latex particles. D₂O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D₂O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D₂O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.     

Human phagocytic cells as oxygen metabolite scavengers

Human neutrophils or monocytes decreased hydrogen peroxide (H₂O₂) concentrationsin vitro. Neutrophils or monocytes decreased H₂O₂ concentrations as well as human erythrocytes. Treatment with aminotriazole or azide decreased both phagocyte and erythrocyte catalase activity and the ability of each cell to decrease H₂O₂ concentrationsin vitro. Prestimulation of phagocytic cells with phorbol myristate acetate (PMA) or opsonized zymosan decreased neither their catalase activity nor their ability to decrease H₂O₂ concentrations. The results suggest that unstimulated or stimulated phagocytic cells can scavenge H₂O₂ and may potentially decrease H₂O₂-mediated tissue injury. The H₂O₂ scavenging potential of phagocytic cells is due at least partially to their catalase activity.This work was supported in part by grants from the National Institutes of Health (P50 HL40784), Johnson and Johnson, Council for Tobacco Research Inc., Procter and Gamble, and Tambrands, Inc.     

Neutrophils may directly synthesize both H2O2 and O2 − since surface stimuli induce their release in stimulus-specific ratios

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O₂ ^– and H₂O₂, the latter being presumed to arise by spontaneous dismutation of the former. If H₂O₂ were indeed derived exclusively from released O₂ ^– according to the equation 2O₂ ^– + 2H⁺ H₂O₂ + O₂, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H₂O₂ should not form when cytochromec is present to scavenge O₂ ^– before it can dismutate. Although H₂O₂ cannot be measured directly in the presence of cytochromec because it is consumed in reoxidizing reduced cytochromec, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochromec. We found that the relative amounts of extracellular H₂O₂ and O₂ ^– that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochromec from H₂O₂ oxidation when some stimuli were used but not with others. For example, the ratio of O₂ ^– to H₂O₂ produced by neutrophils exposed to PMA was about 2:1, the expected result if H₂O₂ were derived from O₂ ^–. However when cytochalasin B was added to the cells before the stimulus, the yield of H₂O₂ was reduced but not the yield of O₂ ^–. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O₂ ^– and H₂O₂. Coating the plastic with IgG doubled cytochromec reduction without effecting H₂O₂. In contrast, coating with albumin reduced H₂O₂ without effecting cytochromec reduction. Soluble IgG aggregates induced production of mostly O₂ ^– whereas immune complexes resulted in release of both metabolites.FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O₂ ^– than H₂O₂. The addition of catalase to the cytochromec solution improved the yield of reduced cytochromec when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H₂O₂ release is not a linear function of the amount of O₂ ^– generated and that either a variable fraction of O₂ ^– spontaneously dismutates to H₂O₂ or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H₂O₂ as well as O₂ ^–. If the latter were true, the pathologic consequences of neutrophil activation would vary depending on whether O₂ ^– was the primary product (chemotactic activation) or whether H₂O₂ was released as well (immune complex stimulation).     

Effect of Recombinant Human Granulocytumacrophage Colony-Stimulating Factor On Neutrophilsuperoxide Production

Abstract

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-a, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and phorbol 12–myristate 13–acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. the results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.     

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.     

Anti-Inflammatory Effect of Lemon Mucilage: In Vivo and In Vitro Studies

The mucilage extracted from a lemon juice centrifugation pulp was studied for its anti-inflammatory effect in rat. In vivo the lemon mucilage significantly inhibited carrageenan-induced edema in rat paw from 59% to 73.5% showing the highest effect at the third hour. In vitro, at the doses of 10^?8, 10^?6, 10^?4 or 10^?2 mg/mL the lemon mucilage stimulated the superoxide anion production in rat testing neutrophils in whole blood but inhibited it in FMLP stimulated cells at the dose of 10^?2 mg/mL. The neutrophils of rats receiving p.o. the lemon mucilage for 21 days showed a significant decrease of 45.5% in O₂^? generation after FMLP stimulation, and a not-significant increase after phorbol-12-myristate-13-acetate (PMA) or zymosan stimulation. Since the activity on zymosan- and PMA-induced O₂^? production was not significant, the inhibition exerted by FMLP in rat neutrophils occurred mainly through the blockade of phospholipase D.     

Endotoxin protection from oxygen toxicity: effect on pulmonary neutrophils and L-selectin

The mechanisms by which sublethal doses of endotoxin protect against hyperoxic lung injury are not completely understood. We hypothesized that endotoxin treatment would result in a decreased inflammatory response to hyperoxia and that this would be accompanied by activation of neutrophils (as evidenced by loss of L-selectin) in the peripheral circulation. Adult rats were injected with endotoxin 0.5 mg/kg prior to and 24 hr after onset of exposure to 98% O₂. After 56 hr of hyperoxia, pulmonary neutrophils were lower in the O₂/endotoxin group compared to O₂ controls as measured by myeloperoxidase in lung homogenates and neutrophil counts in bronchoalveolar lavage fluid. Circulating neutrophils were also significantly lower in the O₂/endotoxin group compared to O₂ controls at 56 hr. Expression of the neutrophil adhesion molecule, L-selectin, was lower at 4 and 24 hr in the endotoxin-treated rats compared to O₂ controls. There were no differences at 48 hr. Expression of CD18 rose significantly in the O₂/endotoxin group after 4 hr, but thereafter did not differ from O₂ controls. In summary, endotoxin protection from O₂ toxicity was associated with reduced neutrophils in the lung and a loss of L-selectin from peripheral blood neutrophils.     

Taxol inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced human neutrophil polarization and H2O2 production while decreasing [3H]FMLP binding

We have studied the effect of taxol on two N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil functions and the possible mechanism by which it inhibits these functions. Taxol inhibited FMLP-induced human neutrophil polarization (a characteristic change in neutrophil shape in response to a chemotactic stimulus) and H₂O₂ generation. Taxol also decreased the specific binding of [³H]FMLP to human neutrophils at 4°C. The decreased binding of FMLP to its receptor may be responsible for the inhibition by taxol of FMLP-induced polarization and H₂O₂ generation.     

Effects of germanium oxide and other chemical compounds on phenylmercury acetate-induced genotoxicity in cultured human lymphocytes

Phenylmercury acetate (PMA), which not only causes an elevation of sister chromatid exchanges (SCEs) but also induces high frequency of endore-duplication in human lymphocytes, may be genotoxic to humans. The major aim of our study was to investigate the effects of germanium oxide (GeO₂), D-penicillamine (D-PA), dimercaprol (BAL), and diltiazem (DTM) on PMA-induced genotoxicity as quantified by SCEs. All concentrations of the four chemical compounds tested alone did not induce genotoxicity in cultured human lymphocytes. However, GeO₂ significantly inhibited PMA-induced genotoxicity in a concentration-dependent manner. Similarly, D-PA at concentrations of 3 μM and 10 μM, and BAL at a concentration of 30 μM produced the antigenotoxic effects. In addition, GeO₂ (1.5 μM) significantly reversed an increase of endoreduplication frequency caused by PMA. In a cell cycle kinetic study, GeO₂ (0.5–5.0 μM) reversed the inhibition of PMA on the proliferating rate index (PRI) of lymphocytes. On the contrary, both D-PA and DTM at concentrations of 30–300 μM markedly potentiated PMA-induced inhibition of PRI. These findings show that GeO₂, D-PA, and BAL could antagonize PMA-induced genotoxicity, and GeO₂ appears to be the most effective. Environ. Mol. Mutagen. 31:157–162, 1998 © 1998 Wiley-Liss, Inc.     

Functional comparison of avian heterophils with human and canine neutrophils

Phagocytosis, intracellular calcium flux, oxidant production and bactericidal activity of chicken heterophils and human and canine neutrophils were compared to assess their functional capabilities with respect to resistance of bacterial infection. Five strains of Staphylococcus were compared for bactericidal susceptiblity; two pathogenic strains isolated from chickens with tenosynovitis, a non-pathogenic strain from the tibiotarsal joint of a normal chicken, a pathogenic strain from the femoral joint of a human and a decapsulated derivative of the human strain. As assayed by flow cytometry, latex spheres were phagocytosed by significantly fewer chicken heterophils than by human or canine neutrophils. Phagocytes from all three species responded to stimulation by autologus serum-opsonised zymosan (aOZ) with an intracellular calcium flux, whereas only human neutrophils responded to N-formyl-methionyl-leucyl-phenylalanine (FMLP). None responded to phorbol myristate acetate (PMA). Neutrophils stimulated with aOZ and PMA produced significantly more H₂O₂ than did heterophils. Human neutrophils produced significantly greater H₂O₂ than did either canine neutrophils or chicken heterophils in response to FMLP. Greater bactericidal activity was observed against the decapsulated human strain and the non-pathogenic avian strain; this increased killing was found to be significant with the canine and avian cells. Avian heterophils were less phagocytic and produced less oxidant in response to zymosan than canine and human neutrophils. In addition, heterophils did not respond to PMA or FMLP.     

Adiponectin inhibits superoxide generation by human neutrophils

Adiponectin (Ad), a member of the adipocytokine family, has been reported to possess antiinflammatory properties. We investigated the effects of full-length human Ad (hAd) on phorbol 12-myristate 13-acetate (PMA)-induced O2-* generation by human neutrophils. hAd, even at the lowest tested concentration of 0.001 microg/ml, after 30-min pretreatment of cells, significantly inhibited O2-* generation by neutrophils stimulated with PMA (100 nM). However, no relation between the dose of hAd and extent of inhibition of PMA-induced O2-* generation was observed with increasing the concentration of hAd up to 1 microg/ml. hAd also significantly inhibited neutrophil O2-* generation stimulated by N-formyl-methionyl-leucyl-phenylalanine (100 microM) and diacylglycerol (500 nM), as well as the PMA-induced neutrophil nitroblue tetrazolium reduction and H2O2 formation. Pretreatment of neutrophils with pronase-digested hAd failed to inhibit the PMA-induced O2-* generation. For the first time, this study revealed that Ad inhibited O2-* generation by neutrophils, possibly through regulation of NADPH oxidase.     

Regulatory Role of Nitric Oxide in Neutrophil Apoptosis

Apoptosis of blood neutrophils from healthy donors was studied under conditions of cell culturing with different concentrations of H₂O₂, selective NO synthase inhibitor, and inductor of NO synthesis (L-arginine). In vitro incubation of neutrophilic leukocytes with 5 mM H₂O₂ led to activation of the apoptotic program in neutrophils, which was seen from increased content of Bax protein in the cells and increased number of apoptotic cells in the culture. Increased content of annexin-positive cells after incubation of neutrophil culture with NO synthase inhibitor suggests involvement of NO in the regulation of neutrophil apoptosis under conditions of oxidative stress, while L-arginine prevented H₂O₂-induced programmed cell death. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 646–650, December, 2008     

Toward in vivo detection of hydrogen peroxide with ultrasound molecular imaging

We present a new class of ultrasound molecular imaging agents that extend upon the design of micromotors that are designed to move through fluids by catalyzing hydrogen peroxide (H₂O₂) and propelling forward by escaping oxygen microbubbles. Micromotor converters require 62 mm of H₂O₂ to move – 1000-fold higher than is expected in vivo. Here, we aim to prove that ultrasound can detect the expelled microbubbles, to determine the minimum H₂O₂ concentration needed for microbubble detection, explore alternate designs to detect the H₂O₂ produced by activated neutrophils and perform preliminary in vivo testing. Oxygen microbubbles were detected by ultrasound at 2.5 mm H₂O₂. Best results were achieved with a 400–500 nm spherical design with alternating surface coatings of catalase and PSS over a silica core. The lowest detection limit of 10–100 μm was achieved when assays were done in plasma. Using this design, we detected the H₂O₂ produced by freshly isolated PMA-activated neutrophils allowing their distinction from naïve neutrophils. Finally, we were also able to show that direct injection of these nanospheres into an abscess in vivo enhanced ultrasound signal only when they contained catalase, and only when injected into an abscess, likely because of the elevated levels of H₂O₂ produced by inflammatory mediators.     

Effect of recombinant human granulocyte/macrophage colony-stimulating factor on neutrophil superoxide production.

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-alpha, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and phorbol 12-myristate 13-acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. The results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.     

Interleukin-1 treatment increases neutrophils but not antioxidant enzyme activity or resistance to ischemia-reperfusion injury in rat kidneys

Hearts from rats treated with interleukin-1 (IL-1) intraperitoneally developed a rapid (6 h after IL-1), transient increase in neutrophils, tissue hydrogen peroxide (H₂O₂), and oxidized glutathione (GSSG) levels, and a subsequent (36 h after IL-1) increase in myocardial glucose-6-phosphate dehydrogenase (G6PD) activity and tolerance to ischemia-reperfusion. In the present investigation, we found that rats treated similarly with IL-1 had increased numbers of neutrophils in their kidneys, which were comparable to myocardial neutrophil increases, but did not develop increased renal tissue H₂O₂ or GSSG levels acutely (6 h after IL-1) or increased G6PD activity or resistance to ischemia-reperfusion injury later (36 h after IL-1). Our findings indicate that IL-1 treatment increased neutrophil accumulation in rat kidneys but did not increase oxidative stress, antioxidant enzyme activity, or resistance to ischemia-reperfusion injury. We conclude that organ-to-organ differences exist with respect to IL-1-induced tolerance.     

Effect of Recombinant Human Granulocytumacrophage Colony-Stimulating Factor On Neutrophilsuperoxide Production

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) induced significant superoxide production in human neutrophils within 30 minutes after addition of stimulus and the response was complete within 2 hr. Other agents known to prime neutrophils, including LPS and tumor necrosis factor-a, lacked activity under the experimental conditions employed. Using a panel of pharmacologic inhibitors, we sought to compare GM-CSF-induced neutrophil superoxide to that produced by cells exposed to N-formyl methionyl-leucyl-phenylalanine (fMet-Leu-Phe) and phorbol 12-myristate 13-acetate (PMA). Each stimulant displayed a different profile. Rolipram, a peak IV phosphodiesterase inhibitor, specifically inhibited neutrophil activation by GM-CSF and fMet-Leu-Phe, while superoxide production stimulated by PMA was unaffected. Staurosporine, a protein kinase C (PK-C) inhibitor, suppressed superoxide production induced by all three neutrophil stimulants. Cytochalasin B totally inhibited superoxide induced by GM-CSF under conditions that promote the fMet-Leu-Phe-induced response. Cytochalasin B did not markedly affect PMA-induced superoxide. the results are consistent with the hypothesis that intact PK-C activity is essential for neutrophil superoxide production, but that differences exist in the initial pathways induced by these neutrophil activators. Superoxide secretion from GM-CSF-treated neutrophils appears to be a direct, delayed response that requires assembly of microfilaments during exposure to the cytokine.     

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H₂O₂, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H₂O₂ was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn²⁺, Fe²⁺, Cu²⁺, and Co²⁺ very markedly enhanced CL induced by mixtures of H₂O₂ and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H₂O₂ probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.     

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on respiratory burst activity of neutrophiis in patients with myelodysplastic syndromes

The superoxide (O₂-releasing capacity in response lo N-fomiyl-methionyl-leucyl-phenylalanine (FMLP)and the priming effects ofrecombinant human granuloeyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage colony-stimulating faclor (rhGM-CSF) on FMLP-induced O₂ release were investigated in neutrophils from 14 patients with myelodysplastic syndromes (MDS). The O₂ -releasing capacity in MDS neutrophils varied from patient to patient. As compared with normal neutJ-ophils. theO₂-releasing capacity in MDS neutrophils was increased in 9/14 patients, nonnal in three patients and decreased in two patients. There was no close relationship between the 02-reIeasing capacity and the peripheral blood neutrophil count or the plasma concentration of C-reactive protein. The priming of neutrophils by rhG-CSF was not observed in five patients, whereas rhGM-CSF primed neutrophils from all patients. The priming eflect of rhGM-CSF was consistently greater than that of rhG-CSF in each patient. The intravenous administration of rhG-CSF (300 μg/body) to two MDS patients showed an increase in the peripheral blood neutrophil count and enhancement of neutrophil O₂ release. These findings demonstrate that the neutrophil O₂-releasing capacity in MDS varies from patient to patient and is not always impaired, and that rhGM-CSF is able to prime neutrophils which never respond to rhG-CSF.     

Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma

We have postulated that murine mammary tumor progression is fueled, in part, by tumor-associated macrophages that deliver sub-lethal oxidative stress to tumor cells. In the present study, we determined whether oxidative stress would affect murine mammary tumor cell attachment to laminin and fibronectin, critical functions in the metastatic process. Sublethal oxidative stress generated by exposure of cells to hydrogen peroxide (H₂O₂, 1–1000 M/L) inhibited tumor cell attachment to immobilized laminin or fibronectin. This oxidant effect was blocked in the presence of catalase which removes H₂O₂. The inhibitory effect on attachment was rapid, with significant inhibition occurring at 5 min; total inhibition was achieved at 60 min with 1 mM H₂O₂. The oxidative stress effect was partially reversible at 20 h post-treatment and occurred at concentrations of H₂O₂ that do not adversely affect cell viability or growth. Pretreatment of tumor cells with H₂O₂ or hypoxanthanine and xanthine oxidase (to generate superoxide radical and H₂O₂) prior to intravenous injection, enhanced experimental lung tumor colony formation. The enhancement of experimental metastatic potential with enzyme-generated oxidative stress was completely reversed by catalase; the H₂O₂-mediated enhancement was only partially reversed with catalase. Thus, treatments that inhibit tumor cell attachment to extracellular matrix proteins in vitro enhance experimental metastasis in vivo.     

Effect of biological surfaces on neutrophil O2- production and its relationship to the CD11b/CD18 integrin-dependent adherence.

The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.